Distinct Functional Roles of beta-Tubulin Isotypes inMicrotubule Arrays of Tetrahymena thermophila, aModel Single-Celled Organism

Background The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2. Methodology/Principal Findings With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically. Conclusion/Significance We conclude that Tetrahymena uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis

Molecular Survey of Toxoplasma gondii in Wild Mammals of Southern Italy

Systematic wildlife surveillance is important to aid the prevention of zoonotic infections that jeopardize human health and undermine biodiversity. Toxoplasma gondii is an opportunistic zoonotic protozoan that can infect all endothermic vertebrates, causing severe disease in immunocompromised humans and cases of congenital transmission. Humans can be infected by ingestion of raw meat containing bradyzoites or water contaminated by oocysts. In our study, we assessed the potential circulation of Toxoplasma gondii in wild mammals by performing surveillance in the Campania region (southern Italy) and surveyed its presence from 2020 to 2022 within the framework of the Regional Plans for Wildlife Surveillance. In detail, 211 individuals belonging to five wild mammals (wolf, fox, wild boar, badger, and roe deer) underwent necropsy and the organs were analyzed by real-time PCR for the detection of the parasite. Toxoplasma gondii was found in 21.8% (46/211) of the subjects examined. No statistically significant differences were noticed between the prevalence and the host’s trophic level or age, rejecting the hypotheses that Toxoplasma gondii will have a higher prevalence in top predators and adult individuals, respectively. Our work emphasized the high circulation of Toxoplasma gondii in wildlife and remarked on the critical role of anthropized areas where domestic cats and wildlife may come into contact, urging a systematic surveillance

Large-scale phylogenomic analysis reveals the phylogenetic position of the problematic taxon Protocruzia and unravels the deep phylogenetic affinities of the ciliate lineages

The Ciliophora is one of the most studied protist lineages because of its important ecological role in the microbial loop. While there is an abundance of molecular data for many ciliate groups, it is commonly limited to the 18S ribosomal RNA locus. There is a paucity of data when it comes to availability of protein-coding genes especially for taxa that do not belong to the class Oligohymenophorea. To address this gap, we have sequenced EST libraries for 11 ciliate species. A supermatrix was constructed for phylogenomic analysis based on 158 genes and 42,158 characters and included 16 ciliates, four dinoflagellates and nine apicomplexans. This is the first multigene-based analysis focusing on the phylum Ciliophora. Our analyses reveal two robust superclades within the Intramacronucleata; one composed of the classes Spirotrichea, Armophorea and Litostomatea (SAL) and another with Colpodea and Oligohymenophorea. Furthermore, we provide corroborative evidence for removing the ambiguous taxon Protocruzia from the class Spirotrichea and placing it as incertae sedis in the phylum Ciliophora

Distinct Functional Roles of β-Tubulin Isotypes in Microtubule Arrays of Tetrahymena thermophila, a Model Single-Celled Organism

<div><h3>Background</h3><p>The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, <em>Tetrahymena thermophila</em>. <em>Tetrahymena</em> forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of <em>Tetrahymena</em> demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (<em>BTU1</em> and <em>BTU2</em>) encode the canonical β-tubulin, BTU2, and six genes (<em>BLT1-6</em>) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.</p> <h3>Methodology/Principal Findings</h3><p>With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins <em>in vivo</em>, we transformed <em>Tetrahymena</em> with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.</p> <h3>Conclusion/Significance</h3><p>We conclude that <em>Tetrahymena</em> uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.</p> </div

The effects of exercise on pain, fatigue, insomnia, and health perceptions in patients with operable advanced stage rectal cancer prior to surgery: a pilot trial

Background: Promoting quality of life (QoL) is a key priority in cancer care. We investigated the hypothesis that, in comparison to usual care, exercise post-neoadjuvant chemoradiation therapy/prior to surgical resection will reduce pain, fatigue, and insomnia, and will improve physical and mental health perceptions in patients with locally advanced stage rectal cancer. Methods: In this non-randomized controlled pilot trial, patients in the supervised exercise group (EG; Mage = 64 years; 64% male) and in the control group (CG; Mage = 72 years; 69% male) completed the European Organization for Research and Treatment of Cancer core Quality of Life questionnaire and the RAND 36-Item Health Survey three times: pre-neoadjuvant chemoradiation therapy (Time 1; nEC = 24; nCG = 11), post-neoadjuvant chemoradiation therapy/pre-exercise intervention (Time 2; nEC = 23; nCG = 10), and post-exercise intervention (Time 3; nEC = 22; nCG = 10). The 6-week exercise intervention was delivered in hospital and comprised of interval aerobic training. Patients trained in pairs three times per week for 30 to 40 minutes. Data were analyzed by Mann-Whitney tests and by Wilcoxon matched-pairs signed rank tests. Results: No significant between-group differences in change were found for any of the outcomes. In both groups, fatigue levels decreased and physical health perceptions increased from pre- to post-exercise intervention. Pain levels also decreased from pre- to post-exercise intervention, albeit not significantly. Conclusions: The findings from this study can be used to guide a more definitive trial as they provide preliminary evidence regarding the potential effects of pre-operative exercise on self-reported pain, fatigue, insomnia, and health perceptions in patients with locally advanced rectal cancer. Trial registration: This study has been registered with clinicaltrials.gov (NCT01325909; March 29, 2011)

Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles

Identification of sortase A (SrtA) substrates in Streptococcus uberis: evidence for an additional hexapeptide (LPXXXD) sorting motif

Sortase (a transamidase) has been shown to be responsible for the covalent attachment of proteins to the bacterial cell wall. Anchoring is effected on secreted proteins containing a specific cell wall motif toward their C-terminus; that for sortase A (SrtA) in Gram-positive bacteria often incorporates the sequence LPXTG. Such surface proteins are often characterized as virulence determinants and play important roles during the establishment and persistence of infection. Intramammary infection with Streptococcus uberis is a common cause of bovine mastitis, which impacts on animal health and welfare and the economics of milk production. Comparison of stringently produced cell wall fractions from S. uberis and an isogenic mutant strain lacking SrtA permitted identification of 9 proteins likely to be covalently anchored at the cell surface. Analysis of these sequences implied the presence of two anchoring motifs for S. uberis, the classical LPXTG motif and an additional LPXXXD motif