Phylogenetic position of Ancyrocephalus (sensu lato) curtus Achmerov, 1952 (Monopisthocotylea, Dactylogyridae), a parasite of fish Perccottus glenii Dybowski, 1877(Gobiiformes: Odontobutidae).

The genus Ancyrocephalus sensu lato is a large assemblage of species of dactylogyrid monopisthocotyleans without clear taxonomic boundaries. Despite an urgent need for revision, only three representatives of this taxon have been molecularly characterised so far. We found specimens of Ancyrocephalus curtus, a previously non-genotyped species, in gills of Perccottus glenii caught in the River Syumnyur, Amur Basin, Russia. The aim of this study was to assess the phylogenetic position of this parasite using partial sequences of 28S rRNA gene. In the phylogenetic tree, A. curtus appeared as a sister taxon to the dactylogyrine genus Gobioecetes. The new molecular evidence supports the hypothesis about the non-monophyletic status of Ancyrocephalus sensu lato.

Mrakia polaris sp. nov. and Mrakia amundsenii sp. nov. (Mrakiaceae, Cystofilobasidiales), two novel psychrophilic yeast species isolated from Arctic habitats.

Four yeast strains belonging to the basidiomycetous yeast genus Mrakia were isolated from diverse habitats in the Ny-Ålesund region (Svalbard, High Arctic): two from vascular plants, one from seawater and one from freshwater. Phylogenetic analysis, based on the ITS region and the D1/D2 domain of the 28S rRNA gene, identified these four strains as representing two novel species within the genus Mrakia. The names Mrakia polaris sp. nov. (MycoBank number: MB 852063) and Mrakia amundsenii sp. nov. (MycoBank number: MB 852064) are proposed. These two new species show distinct psychrophilic adaptations, as they exhibit optimal growth at temperatures between 10 and 15°C, while being unable to grow at 25°C. The holotype of M. polaris sp. nov. is CPCC 300345T, and the holotype of M. amundsenii sp. nov. is CPCC 300572T.

HMGB1 prefers to interact with structural RNAs and regulates rRNA methylation modification and translation in HeLa cells.

BACKGROUND: High-mobility group B1 (HMGB1) is both a DNA binding nuclear factor modulating transcription and a crucial cytokine that mediates the response to both infectious and noninfectious inflammation such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. HMGB1 has been proposed to control ribosome biogenesis, similar as the other members of a class of HMGB proteins. RESULTS: Here, we report that HMGB1 selectively promotes transcription of genes involved in the regulation of transcription, osteoclast differentiation and apoptotic process. Improved RNA immunoprecipitation by UV cross-linking and deep sequencing (iRIP-seq) experiment revealed that HMGB1 selectively bound to mRNAs functioning not only in signal transduction and gene expression, but also in axon guidance, focal adhesion, and extracellular matrix organization. Importantly, HMGB1-bound reads were strongly enriched in specific structured RNAs, including the domain II of 28S rRNA, H/ACA box snoRNAs including snoRNA63 and scaRNAs. RTL-P experiment showed that overexpression of HMGB1 led to a decreased methylation modification of 28S rRNA at position Am2388, Cm2409, and Gm2411. We further showed that HMGB1 overexpression increased ribosome RNA expression levels and enhanced protein synthesis. CONCLUSION: Taken together, our results support a model in which HMGB1 binds to multiple RNA species in human cancer cells, which could at least partially contribute to HMGB1-modulated rRNA modification, protein synthesis function of ribosomes, and differential gene expression including rRNA genes. These findings provide additional mechanistic clues to HMGB1 functions in cancers and cell differentiation.

A cyst-forming coccidian with large geographical range infecting forest and commensal rodents: Sarcocystis muricoelognathis sp. nov.

BACKGROUND: The geographic distribution and host-parasite interaction networks of Sarcocystis spp. in small mammals in eastern Asia remain incompletely known. METHODS: Experimental infections, morphological and molecular characterizations were used for discrimination of a new Sarcocystis species isolated from colubrid snakes and small mammals collected in Thailand, Borneo and China. RESULTS: We identified a new species, Sarcocystis muricoelognathis sp. nov., that features a relatively wide geographic distribution and infects both commensal and forest-inhabiting intermediate hosts. Sarcocystis sporocysts collected from rat snakes (Coelognathus radiatus, C. flavolineatus) in Thailand induced development of sarcocysts in experimental SD rats showing a type 10a cyst wall ultrastructure that was identical with those found in Rattus norvegicus from China and the forest rat Maxomys whiteheadi in Borneo. Its cystozoites had equal sizes in all intermediate hosts and locations, while sporocysts and cystozoites were distinct from other Sarcocystis species. Partial 28S rRNA sequences of S. muricoelognathis from M. whiteheadi were largely identical to those from R. norvegicus in China but distinct from newly sequenced Sarcocystis zuoi. The phylogeny of the nuclear 18S rRNA gene placed S. muricoelognathis within the so-called S. zuoi complex, including Sarcocystis attenuati, S. kani, S. scandentiborneensis and S. zuoi, while the latter clustered with the new species. However, the phylogeny of the ITS1-region confirmed the distinction between S. muricoelognathis and S. zuoi. Moreover, all three gene trees suggested that an isolate previously addressed as S. zuoi from Thailand (KU341120) is conspecific with S. muricoelognathis. Partial mitochondrial cox1 sequences of S. muricoelognathis were almost identical with those from other members of the group suggesting a shared, recent ancestry. Additionally, we isolated two partial 28S rRNA Sarcocystis sequences from Low's squirrel Sundasciurus lowii that clustered with those of S. scandentiborneensis from treeshews. CONCLUSIONS: Our results provide strong evidence of broad geographic distributions of rodent-associated Sarcocystis and host shifts between commensal and forest small mammal species, even if the known host associations remain likely only snapshots of the true associations.

Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study.

BACKGROUND: HIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25-30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa. METHODS: We developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018-21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis. FINDINGS: When compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1-99·5) and of the QSP1 assay was 90·4% (85·2-94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55-75) and 68% (57-73), respectively, and lower C gattii rates of 21% (14-31) and 8% (4-14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture). INTERPRETATION: QSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear. FUNDING: European and Developing Countries Clinical Trials Partnership; Swedish International Development Cooperation Agency; Wellcome Trust/UK Medical Research Council/UKAID Joint Global Health Trials; and UK National Institute for Health Research.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

Revisiting the phylogeny of the family Miridae (Heteroptera: Cimicomorpha), with updated insights into its origin and life history evolution.

Heteroptera is one of the most successfully adapted groups on Earth and can be observed in almost every environment. Within the evolution of heteropteran insects, Miridae show remarkable diversity (>11,700 spp.), accounting for a quarter of all Heteroptera. However, their phylogeny is still unclear, and no plausible theory for the driving force of their diversification has been established. In this work, we provide new suggestions for the phylogeny of Miridae using a larger dataset than previous studies. In addition, we suggest an alternative evolutionary history based on newly calibrated divergence dates for Miridae and its subordinate groups, and present probable factors of the family's success in terms of species diversity. The entire dataset comprises 16 outgroups and 188 ingroup taxa including all seven known subfamilies and 37 out of 45 known tribes. Each species is aligned as 3,577 bp with six molecular loci (COI, 16S rRNA, 18S rRNA, 28S rRNA D3 region, H2A, and H3A). Among the molecular markers, we are the first to test histone genes (H2A, H3A) in Miridae. Our results raise the following points about phylogenetic relationships: i) The earliest group to diverge from Miridae was Monaloniini (Bryocorinae). ii) Bryocorinae and Cylapinae are polyphyletic, Deraeocorinae and Orthotylinae also rendered as non-monophyletic group. iii) Termatophylini and Coridromiini separated from Deraeocorinae and Orthotylinae respectively. iv) Four large tribes, Orthotylini, Phylini, Deraeocorini and Mirini are non-monophyletic. The results from our ancestral state reconstruction and divergence date estimation suggest the following: i) Miridae first diverged during the Late Jurassic (approx. 163.4 Mya), and the divergence dates of most subfamilies and tribes overlap with angiosperm radiation, which perhaps synergized their diversification. ii) Ancestral reconstruction results for Miridae reveal it to be predominantly phytophagous and diverge to oligophagy mainly in plant-tissue habitats, which could have allowed the mirids to select optimal tactics as plant-dwellers. iii) The common ancestor of Miridae originated among plant-dwellers mainly on Eudicots, and that tendency was largely maintained, but sporadic host shifts also occurred.

Characterization of a new IN-I-PpoI fusion protein and a homology-arm containing transgene cassette that improve transgene expression persistence and 28S rRNA gene-targeted insertion of lentiviral vectors.

Targeting transgene integration into a safe genomic locus would be very important for gene therapy. We have generated lentivirus vectors containing the ribosomal RNA-recognising I-PpoI endonuclease fused to viral integrase, and transgene cassettes with target site homology arms to enhance insertion targeting. These new vectors were characterised with respect to the persistence of transgene expression, insertion targeting efficiency and chromosomal integrity of the transduced cells. The aim was to find an optimally safe and effective vector for human gene therapy. Fusion protein vectors with high endonuclease activity were the most effective in the accurate targeting of transgene insertion. The homology construct increased the insertion targeting efficiency to 28% in MRC-5 cells. However, karyotyping analysis showed that the high endonuclease activity induced the formation of derivative chromosomes in as many as 24% of the analysed primary T lymphocytes. The persistence of transgene expression was excellent in homology arm-containing fusion protein vectors with reduced endonuclease activity, and these fusion proteins did not cause any detectable chromosomal rearrangements attributable to the endonuclease activity. We thus conclude that instead of the fusion protein vectors that carry a highly active endonuclease, our vectors with the ability to tether the lentivirus preintegration complex to benign loci in the genome without high ribosomal DNA cleavage activity are better suited for lentivirus-based gene therapy applications.

Mechanism for inhibition of cytotoxicity of Shiga toxin by luteolin.

Enterohemorrhagic or Shiga toxin-producing Escherichia coli is a food-poisoning bacterium that grows in the intestine to produce Shiga toxin (Stx). In this study, the effects of 20 polyphenols on the cytotoxicity of Stx1 and Stx2 in Vero cells were investigated. Among these, epigallocatechin gallate, butein, isorhapontigenin, hesperetin, morin, luteolin, resveratrol, and rhapontigenin showed inhibitory effects on the cytotoxicity of Stxs at 0.4 mmol/L. Furthermore, Vero cells pre-treated with these polyphenols were resistant to Stx at 0.4 mmol/L. However, luteolin showed the most potent inhibitory and cytoprotective effect against Stxs at 0.08 mmol/L or more. This inhibitory mechanism of luteolin was determined using a cell-free protein synthesis system and quantitative reverse transcription PCR assay to detect depurination of 28S rRNA in Vero cells. Luteolin did not inhibit the cell-free protein synthesis by Stxs, suggesting that the enzymatic activity of the Stx A subunit was not inhibited by luteolin. The depurination of 28S rRNA by Stxs was also investigated in Vero cells. The 28S rRNA depurination by Stxs was suppressed in Vero cells treated with Stxs which had been pretreated with luteolin. These results suggest that luteolin inhibits the incorporation of Stxs into Vero cells. This is the first report to show that luteolin inhibits the cytotoxicity of both Stx1 and Stx2 by inhibiting the incorporation of Stxs into Vero cells.

SNORD88C guided 2'-O-methylation of 28S rRNA regulates SCD1 translation to inhibit autophagy and promote growth and metastasis in non-small cell lung cancer.

Small nucleolar RNAs (snoRNAs) have been shown to play critical regulatory roles in cancer development. SNORD88C, which located at the intronic region of C19orf48 in chromosome 19q.33 with a 97-nt length was screened through database and snoRNA-sequencing. We firstly verified this snoRNA was up-regulated in tissue and plasma and served as a non-invasive diagnostic biomarker; then confirmed that SNORD88C promoted proliferation and metastasis of NSCLC in vitro and in vivo. Mechanistically, SNORD88C promoted 2'-O-methylation modification at the C3680 site on 28S rRNA and in turn enhanced downstream SCD1 translation, a central lipogenic enzyme for the synthesis of MUFA that can inhibit autophagy by regulating lipid peroxidation and mTOR, providing the novel insight into the regulation of SNORD88C in NSCLC.

28S rRNA-Derived Fragments Represent an Independent Molecular Predictor of Short-Term Relapse in Prostate Cancer.

Prostate cancer (PCa) is a global health concern, being a leading cause of cancer-related mortality among males. Early detection and accurate prognosis are crucial for effective management. This study delves into the diagnostic and prognostic potential of 28S rRNA-derived fragments (rRFs) in PCa. Total RNA extracted from 89 PCa and 53 benign prostate hyperplasia (BPH) tissue specimens. After 3'-end polyadenylation, we performed reverse transcription to create first-strand cDNA. Using an in-house quantitative real-time PCR (qPCR) assay, we quantified 28S rRF levels. Post-treatment biochemical relapse served as the clinical endpoint event for survival analysis, which we validated internally through bootstrap analysis. Our results revealed downregulated 28S rRF levels in PCa compared to BPH patients. Additionally, we observed a significant positive correlation between 28S rRF levels and higher Gleason scores and tumor stages. Furthermore, PCa patients with elevated 28S rRF expression had a significantly higher risk of post-treatment disease relapse independently of clinicopathological data. In conclusion, our study demonstrates, for the first time, the prognostic value of 28S rRF in prostate adenocarcinoma. Elevated 28S rRF levels independently predict short-term PCa relapse and enhance risk stratification. This establishes 28S rRF as a potential novel molecular marker for PCa prognosis.

Remarks on phylogeny and molecular variations of criconematid species (Nematoda: Criconematidae) with case studies from Vietnam.

The family Criconematidae is a remarkable group of nematodes, containing roughly 600 nominal root-ectoparasitic species, of which many species are known to be significant agricultural pests. Strikingly, our phylogenetic analyses based on 18S, D2-D3 of 28S rRNA, and COI mtDNA sequences of criconematid species, supported by tree topology tests (SH and AU tests), revealed that almost all studied genera, including Criconema, Ogma, Crossonema, Discocriconema, Hemicriconemoides, Criconemoides, Mesocriconema, and Lobocriconema, are not monophyletic groups, a finding that is partly contrary to those of previous studies on these groups. Our results suggest that key morphological characters used in the classification of Criconematidae are the consequence of convergent evolution. It is clear from our studies that the species status of at least 40 sequences of criconematid species from GenBank must be either revised or reconsidered, with analyses based on a polyphasic approach that includes different tree- and distance-based molecular species-delimitation methods (bPTP, GMYC, ABGD1, and ABGD2). Our studies found the ABGD2 output of the automatic barcode method to agree remarkably well with established species delimitations, while in general, the four species-delimitation results corresponding to three barcode regions forwarded significantly more putative species compared to those originally considered. This study also characterised for the first time the populations of Criconemoides myungsugae and Discocriconemella hensungica associated with Vietnamese ginseng, one of the most precious and rare ginseng varieties in the world. Although these populations are morphologically in agreement with the original descriptions of C. myungsugae and D. hengsungica, their molecular data display notable variations compared to the sequences deposited in GenBank. These species demonstrate clearly the immense molecular variations that can be observed in several species of the family Criconematidae.

Human NOP2/NSUN1 regulates ribosome biogenesis through non-catalytic complex formation with box C/D snoRNPs.

5-Methylcytosine (m5C) is a base modification broadly found on various RNAs in the human transcriptome. In eukaryotes, m5C is catalyzed by enzymes of the NSUN family composed of seven human members (NSUN1-7). NOP2/NSUN1 has been primarily characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S ribosomal RNA (rRNA). Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions and substrates remain poorly characterized. Here, we used a miCLIP-seq approach to identify human NOP2/NSUN1 RNA substrates. Our analysis revealed that NOP2/NSUN1 catalyzes the deposition of m5C at position 4447 on the 28S rRNA. We also find that NOP2/NSUN1 binds to the 5'ETS region of the pre-rRNA transcript and regulates pre-rRNA processing through non-catalytic complex formation with box C/D snoRNAs. We provide evidence that NOP2/NSUN1 facilitates the recruitment of U3 and U8 snoRNAs to pre-90S ribosomal particles and their stable assembly into snoRNP complexes. Remarkably, expression of both WT and catalytically inactive NOP2/NSUN1 in knockdown background rescues the rRNA processing defects and the stable assembly of box C/D snoRNP complexes, suggesting that NOP2/NSUN1-mediated deposition of m5C on rRNA is not required for ribosome synthesis.

Analysis of the Sequence Preference of Saporin by Deep Sequencing.

Ribosome-inactivating proteins (RIPs) are RNA:adenosine glycosidases that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin loop (SRL) in 28S rRNA. The GAGA sequence at the top of the SRL or at the top of a hairpin loop is assumed to be their target motif. Saporin is a RIP widely used to develop immunotoxins for research and medical applications, but its sequence specificity has not been investigated. Here, we combine the conventional aniline cleavage assay for depurinated nucleic acids with high-throughput sequencing to study sequence-specific depurination of oligonucleotides caused by saporin. Our data reveal the sequence preference of saporin for different substrates and show that the GAGA motif is not efficiently targeted by this protein, neither in RNA nor in DNA. Instead, a preference of saporin for certain hairpin DNAs was observed. The observed sequence-specific activity of saporin may be relevant to antiviral or apoptosis-inducing effects of RIPs. The developed method could also be useful for studying the sequence specificity of depurination by other RIPs or enzymes.

Four carangid fish species as new host records for Kudoa trachuri Matsukane, Sato, Tanaka, Kamata et Sugita-Konishi, 2011 (Myxozoa: Multivalvulida), and description of a new species, Kudoa longichorda sp. n., forming pseudocysts in the muscle of Decapterus tabl Berry.

Multivalvulid myxosporeans of the genera Kudoa Meglitsch, 1947 and Unicapsula Davis, 1924 (Cnidaria: Myxozoa) are often the cause of unsightly cyst formation or postmortem myoliquefaction in the trunk muscle of commercial marine fish, which reduces the market value of infected individuals. Twenty species (18 Kudoa spp. and two Unicapsula spp.) have been recorded from carangid fish, although the majority of them, excluding polyxenous species, such as K. amamiensis Egusa et Nakajima, 1980, K. iwatai Egusa et Shiomitsu, 1983, K. nova Naidenova, 1975, K. quadratum (Thélohan, 1895) and K. yasunagai (Hsieh et Chen, 1984), are limited to a single or a few fish species. We report the occurrence of macroscopic cysts of Kudoa trachuri Matsukane, Sato, Tanaka, Kamata et Sugita-Konishi, 2011 in the trunk muscle of four new host fish species, i.e., Pseudocaranx dentex (Bloch et Schneider), Decapterus akaadsi Abe, D. muroadsi (Temminck et Schlegel) and Decapterus tabl Berry, fished from the Philippine Sea (Northwest Pacific Ocean), off southwestern of Japan. Myxospore morphology and genetic characteristics of the ribosomal RNA gene (rDNA) of these isolates were consistent with previous records of K. trachuri from Trachurus japonicus (Temminck et Schlegel) from around Japan. In addition, a new species of Kudoa that forms long filamentous pseudocysts in trunk myofibres was found in four of the six D. tabl collected in this study. We describe Kudoa longichorda sp. n. for this new isolate, based on its morphology of subquadrate myxospores with four shell valves and polar capsules and with small dimensions (length 4.3-5.5 µm, width 6.0-6.8 µm, thickness 4.8-6.3 µm, polar capsule length 2.3-3.1 µm, polar capsule width 1.1-1.7 µm), as well as 18S and 28S rDNA sequences distinct from those of known species.

Influence of oestrogen on satellite cells and myonuclear domain size in skeletal muscles following resistance exercise.

BACKGROUND: Oestrogen deficiency reduces skeletal muscle mass and force generation in postmenopausal women. Muscle mass is maintained by satellite cells, which are regulated by oestrogen. Although oestrogen therapy enhances muscle hypertrophy induced by resistance training in postmenopausal women, the molecular mechanism is unclear. METHODS: Adult female rats (10 weeks old) were divided into six groups: sham sedentary (Sham-Sed), sham climbing training (Sham-CT), ovariectomy sedentary (OVX-Sed), ovariectomy climbing training (OVX-CT), ovariectomy plus oestrogen treatment sedentary (OVX+E-Sed), and ovariectomy plus oestrogen treatment climbing training (OVX+E-CT). At 8 weeks after ovariectomy, rats in the training group were trained (one session every 3 days for 8 weeks) to climb a ladder while bearing a load. Oestrogen treatment involved subcutaneous insertion of a 17ß-oestradiol pellet. After 8 weeks, the flexor hallucis longus muscle was collected and analysed. RESULTS: Following climbing training, the flexor hallucis longus muscle mass and muscle-to-body weight ratios were dramatically increased by training (main effect of training, P < 0.01); the OVX+E-CT group showed the highest values (main effect of group, P < 0.01). The cross-sectional area of all muscle fibre types was increased by training (main effect of training, P < 0.01). Particularly, the cross-sectional area of MHC IIa in the OVX+E-CT group was significantly larger than that in the Sham-CT and OVX-CT groups. Satellite cell numbers were increased in all training groups (main effect of training, P < 0.05), and the myonuclear number was increased by training (main effect of training, P < 0.01), but there was no main group effect. The myonuclear domain size of all muscle fibre types and MHC IIa was increased in all training groups (main effect of training, P < 0.01) and showed a main group effect (P < 0.01). The myonuclear domain sizes of all muscle fibre types and MHC IIa in the OVX+E-CT group were significantly larger than those in the Sham-CT and OVX-CT groups. The total RNA contents revealed main effects of training and the group (P < 0.01); the OVX+E-CT group showed the highest contents (main effect of group, P < 0.01). The mRNA and protein levels of rpS6 were increased in the OVX+E-Sed and CT groups (main effects of group, P < 0.05). Particularly, the 28S ribosomal RNA content in OVX+E-Sed group was significantly higher than that in the OVX-Sed group. CONCLUSIONS: Oestrogen enhanced the resistance training-induced increase in myonuclear domain size but did not affect satellite cells and ribosome biogenesis.

A phylogenetic overview of Squamanita, with descriptions of nine new species and four new combinations.

Nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) sequence data from eight type specimens of previously described Squamanita species were obtained. Phylogenetic analysis of ITS and partial nuc 28S rDNA data revealed Squamanita as paraphyletic splitting into two monophyletic groups, which we recognize as the genera Squamanita and Dissoderma. We accept 14 Squamanita and nine Dissoderma species, provide the first sequences of 13 of these, and describe six new species of Squamanita and three new species of Dissoderma. We transfer three species of Squamanita into Dissoderma, one into Cystoderma, and treat S. basii and S. umbilicata as synonyms of D. paradoxum. Squamanita can be distinguished from Dissoderma by the generally larger fleshier basidiomata with a tricholomatoid or amanitoid stature and yellowish to tawny brown pileus and often similarly colored stipe. Most species have cheilo- and pleurocystidia. Species of Dissoderma are small, collybioid or mycenoid, lack cystidia, and the pileus and often upper stipe are purplish gray. Both genera parasitize basidiomata of other agarics.

Is Gymnophallus Odhner, 1900 (Trematoda: Gymnophallidae) polyphyletic? A new hypothesis based on phylogenetic position of Gymnophallus deliciosus (Olsson, 1893).

Gymnophallus deliciosus is a type species of the genus Gymnophallus. We collected this trematode species from gallbladder of Larus argentatus caught in the White Sea (Kandalaksha Bay, Chupa Inlet) and obtained the sequences of its nuclear 18S and 28S rDNA genes. Our recently obtained phylogenetic data support a sister group relationship of this species with G. choledochus. However, other species of the genus Gymnophallus-G. australis (KM246854) and G. minutus (KM268111)-do not branch with the group G. choledochus + G. deliciosus or with each other. Our study revealed that the genus Gynmnophallus is probably a polyphyletic taxon, and the genus affiliation of its representatives should be re-examined in future.

First elucidation of a didymozoid life cycle: Saccularina magnacetabula n. gen. n. sp. infecting an arcid bivalve.

The first first-intermediate host for a species of Didymozoidae (Trematoda: Hemiuroidea), a bivalve of the family Arcidae, is identified using multi-loci molecular data. First intermediate, (likely) third intermediate, and adult stages of a new didymozoid taxon (Saccularina magnacetabula n. gen. n. sp.) from Moreton Bay, Queensland, Australia were collected from the Sydney cockle Anadara trapezia (Deshayes) (Arcoidea: Arcidae), Sillago sp. (Sillaginidae) and Elops hawaiensis Regan (Elopiformes: Elopidae), respectively, and genetically matched. Infections in A. trapezia were present as sporocysts and cystophorous cercariae, and infected tissue at the base of the gills. Morphologically, S. magnacetabula is distinctive relative to all other didymozoids in the combination of hermaphroditism, mate-pairing, filiform body shape, the presence of a ventral sucker, a single testis, and a saccular excretory vesicle at the posterior extremity. Molecular sequence data were generated for S. magnacetabula and 42 other putative didymozoid species to explore relationships within the Didymozoidae and Hemiuroidea. In molecular phylogenetic analyses of the 28S rDNA region, the new genus forms a clade with an undescribed taxon from the redthroat emperor, Lethrinus miniatus (Bloch & Schneider) (Perciformes: Lethrinidae), from the Great Barrier Reef, and another uncharacterised taxon from E. hawaiensis. This clade is sister to a moderately well-supported clade comprising all other didymozoid species for which sequences are available, including representatives of five of the six presently recognised subfamilies. The infection of a bivalve by a didymozoid is discussed in the context of the overwhelming use of gastropod molluscs as first intermediate hosts by the Hemiuroidea.

Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins.

During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin-ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (VHHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such VHHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these VHHs block RTA-P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as "intrabodies," these VHHs rendered cells resistant to ricin intoxication. One VHH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation.