Post-acute cardiac complications following SARS-CoV-2 infection in partial lipodystrophy due to LMNA gene p.R349W mutation.

PURPOSE: SARS-CoV-2 infection may cause varying degrees of cardiac injury and the presence of underlying cardiovascular morbidities contributes to the frequency and severity of occurrence of this complication. Lipodystrophy syndromes are frequently characterized by severe metabolic derangements that represent relevant cardiovascular risk factors. Besides causing lipodystrophy, mutations in the lamin A/C (LMNA) gene can lead to a wide spectrum of tissue-specific disorders including cardiac involvement. METHODS AND RESULTS: We herein examine the case of two patients affected by atypical progeroid syndrome and partial lipodystrophy due to a heterozygous missense LMNA mutation c.1045 C > T (p.R349W) who presented initially with mild COVID-19 and developed severe cardiovascular complications within few weeks of SARS-CoV-2 infection. Before being infected with SARS-CoV-2, our patients had cardiovascular morbidities (mild mitral regurgitation in one patient, ischemic heart disease with bifascicular block in the other patient) in adjunct to cardiovascular risk factors, but the SARS-CoV-2 infection contributed to quickly and significantly decompensate their balance. CONCLUSION: These findings warn that patients affected by LMNA p.R349W mutation and likely other LMNA mutations associated with cardiovascular morbidity should be considered at extremely elevated risk of post-acute cardiological manifestations and should therefore undergo a vigilant follow-up after SARS-CoV-2 infection. Both patients developed COVID-19 before the specific vaccination was available to them and this unfortunate situation should remark the importance of vaccination coverage against SARS-CoV-2 infection for all patients affected by lipodystrophy, especially those with underlying comorbidities.

Epigenetic Changes in Chronic Inflammatory Diseases.

The number of people diagnosed with chronic inflammatory diseases has increased noteworthy in the last 40 years. Spondyloarthritis (SpA), inflammatory bowel diseases (IBD), and psoriasis are the most frequent chronic inflammatory diseases, resulting from a combination of genetic predisposition and environmental factors. Epigenetic modifications include DNA methylation, histone modifications, and small and long noncoding RNAs. They are influenced by environmental exposure, life-style, and aging and have recently been shown to be altered in many complex diseases including inflammatory diseases. While epigenetic modifications have been well characterized in other diseases such as cancer and autoimmune diseases, knowledge on changes in inflammatory diseases is lagging behind with some disease-specific differences. While the DNA methylation profile of different cell types in patients with IBD has been relatively well described, less is known on changes implicated in psoriasis, and no systematic genome-wide studies have so far been performed in SpA. In this chapter, we review in detail the reported changes in patterns of DNA methylation and posttranslational histone modifications in chronic inflammatory diseases highlighting potential connections between disease-associated pathophysiological changes such as the dysbiosis of the microbiome or genetic variations associated with disease susceptibility and the epigenome. We also discuss important parameters of meaningful epigenetic studies such as the use of well defined, disease-relevant cell populations, and elude on the potential future of engineering of the epigenome in inflammatory diseases.

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.

Cell cycle inhibition therapy that targets stathmin in in vitro and in vivo models of breast cancer.

Stathmin is the founding member of a family of microtubule-destabilizing proteins that have a critical role in the regulation of mitosis. Stathmin is expressed at high levels in breast cancer and its overexpression is linked to disease progression. Although there is a large body of evidence to support a role for stathmin in breast cancer progression, the validity of stathmin as a viable therapeutic target for breast cancer has not been investigated. Here, we used a bicistronic adenoviral vector that co-expresses green fluorescent protein and a ribozyme that targets stathmin messenger RNA in preclinical breast cancer models with different estrogen receptor (ER) status. We examined the effects of anti-stathmin ribozyme on the malignant phenotype of breast cancer cells in vitro and in xenograft models in vivo both as a single agent and in combination with chemotherapeutic agents. Adenovirus-mediated gene transfer of anti-stathmin ribozyme resulted in a dose-dependent inhibition of proliferation and clonogenicity associated with a G2/M arrest and increase in apoptosis in both ER-positive and ER-negative breast cancer cell lines. This inhibition was markedly enhanced when stathmin-inhibited breast cancer cells were exposed to low concentrations of taxol, which resulted in virtually complete loss of the malignant phenotype. Interestingly, breast cancer xenografts treated with low doses of anti-stathmin therapy and taxol showed regression in a majority of tumors, while some tumors stopped growing completely. In contrast, combination of anti-stathmin ribozyme and adriamycin resulted in only a modest inhibition of growth in vitro and in breast cancer xenografts in vivo. Although inhibition of tumor growth was observed in both the combination treatment groups compared with groups treated with single agent alone, combination of anti-stathmin therapy and taxol had a more profound inhibition of tumorigenicity, as both agents target the microtubule pathway. Clinically, these findings are highly relevant because taxol is one of the most active chemotherapeutic agents in breast cancer. These studies provide the proof-of-principle that stathmin provides an attractive molecular target, which could serve as a primary focus of novel approaches to breast cancer.

Tumour necrosis factor-alpha blockers in rheumatoid arthritis: review of the clinical experience.

Tumour necrosis factor (TNF)-alpha has been found to play a central role in the pathogenesis of rheumatoid arthritis, leading to development of novel drug therapies that neutralise the deleterious effects of this cytokine. This new concept of immunobiological treatment of rheumatoid arthritis has yielded successful results. Although the 2 currently available TNFalpha blockers, infliximab and etanercept, differ in structure, mechanism of action and pharmacokinetics, they have provided similar benefits both in clinical improvement and in slowing and even arresting the progression of radiographic damage. This therapeutic response seems to be unequalled by "conventional" treatments in rheumatoid arthritis, and is incontestably a turning point in the therapeutic management of this disease.

Divergence between two Antarctic species of the ciliate Euplotes, E. focardii and E. nobilii, in the expression of heat-shock protein 70 genes.

Most organisms oppose many environmental stresses by rapidly enhancing synthesis of the highly conserved Hsp70 family of heat-shock proteins. Two ciliates which are endemic in Antarctic coastal seawater, Euplotes focardii and E. nobilii, and behave as psychrophile and psychrotroph micro-organisms, respectively, revealed a divergence in the capacity to respond to thermal stress with an activation of the transcription of their hsp70 genes. In both species, these genes were shown to be represented by thousands of copies in the cell's somatic functional nucleus (macronucleus). However, while a strong transcriptional activity of hsp70 genes was induced in E. nobilii cells transferred from 4 to 20 degrees C, a much smaller increase was revealed in heat-shocked cells of E. focardii. These findings suggest a closer adaptation to the stably cold Antarctic waters in the genetic response of E. focardii to thermal stress.

Novelty in phylogeny of gastrotricha: evidence from 18S rRNA gene.

Gastrotricha form a phylum which is crucial for defining the origin of pseudocoelomates, in that they share a number of characters with Rotifera and Nematoda but also with acoelomates, and even the evolutionary relationships within the phylum are anything but defined. For this reason the first extensive molecular data on Gastrotricha from the 18S rRNA sequences of both orders have been obtained and analyzed. Sequence analyses show that the phylum Gastrotricha is strictly monophyletic along an evolutionary line quite distinct from that of both Rotifera and Nematoda. A new view of the evolutionary history of the phylum Gastrotricha is put forward, in which Chaetonotida, and not Macrodasyida, are the most primitive forms of the group, contrary to the commonly held view. A polyphyletic origin of aschelminthes is supported, and the misleading term pseudocoelomates should be discarded.

Molecular cloning of the gene encoding an acidic ribosomal protein of the P2 family from the ciliate Euplotes raikovi.

We have characterized a macronuclear gene of the ciliate protozoan Euplotes raikovi, which encodes an acidic ribosomal protein of the P protein family. This gene shows the typical organization of the hypotrich ciliate macronuclear "gene-sized" molecules with Euplotes telomeres at the ends. The longest open reading frame encodes a conceptual protein of 113 amino acid residues, with a molecular mass and pI value of 11.45 kDa and 3.97, respectively. By using sequence homology analysis, the protein was found to belong to the ribosomal P2 protein family and was named Er P2, where Er stands for Euplotes raikovi. These proteins, generally called A (acidic/alanine rich) proteins in prokaryotes and P (phosphorylated) proteins in eukaryotes, in which they are divided into P1 and P2 families, play a role in the elongation step of protein synthesis. Approximately 40% amino acid sequence identity was found between the cloned protein and other known protozoan ribosomal P2 proteins. Within its N-terminal half, this protein contains several potential kinase phosphorylation sites. Protein Er P2 differs markedly from the consensus P protein sequence in its C-terminal region, usually highly conserved among eukaryotic ribosomal P proteins, and shows similarities with the C-terminus of the archaebacterial ribosomal A proteins. To our knowledge, this E. raikovi protein represents the first demonstration of a ribosome-associated protein of the P2 family in a ciliate protozoan.

Cadmium metallothionein gene of Tetrahymena pyriformis.

A genomic sequence from Tetrahymena pyriformis, encoding a cadmium-induced metallothionein has been cloned. The gene encodes a transcript of 487 bases, with an intronless coding region of 324 nt, using TGA as the stop codon, TAA coding for glutamine, and the translational initiation sequence AAAATGG. Two regions of internal similarity in the coding sequence support the hypothesis that the Tetrahymena protein arose by gene duplication. The sequences of untranslated regions show some similarities with nematode MT-1 and MT-2 transcripts. Sequence of 525 bases upstream of the transcription start contains a TATA box, a CAAT box reverse complement, and many short stretches partially matching the AP-1 and ACE-1 binding sites, but no characteristic sequences found in other metallothionein promoters.

Development of the Abbott AxSYM Free PSA assay: performance characteristics and preliminary clinical evaluation.

The AxSYM Free PSA assay was demonstrated to have good analytical sensitivity and reproducibility. The F/T ratio determinations for 385 men tested during the Prostate Awareness Week who had biopsies due to an elevated total PSA value and/or a suspicious DRE demonstrated that the percentage of free PSA was lower in patients found to have prostate cancer than those that were biopsy negative for the overall group and for all patient categories examined. The optimal strategy for combining PSA values, F/T ratios, DRE and other clinical and diagnostic parameters to improve the early detection of prostate cancer requires additional clinical studies.

[Late cerebral radionecrosis of cystic aspect]. / Radionécroses cérébrales tardives d'aspect kystique.

Two cases of delayed cerebral radionecrosis with cystic presentation are reported. The patients had received radiation therapy for a cutaneous tumor 16 and 12 years before. Surgical extirpation of the cerebral lesion permitted to confirm the diagnosis and to treat the patients with success. A cystic aspect has been reported rarely in the neuroradiologic descriptions of the cerebral radionecrosis in the literature. Its mechanism is discussed. The treatment of the delayed cerebral radionecrosis is not clearly established, but a cystic presentation is an argument for surgery.

Cold-adapted microtubules: characterization of tubulin posttranslational modifications in the Antarctic ciliate Euplotes focardii.

In cold poikilotherm organisms, microtubule assembly is promoted at temperatures below 4 degrees C and cold-induced depolymerization is prevented. On the basis of the results of investigations on cold-adapted fishes, the property of cold adaptation is ascribed to intrinsic characteristics of the tubulins. To fully understand cold adaptation, we studied the tubulins of Euplotes focardii, an Antarctic ciliated protozoan adapted to temperatures ranging from -2 to +4 degrees C. In this organism, we had previously sequenced one beta-tubulin gene and, then identified three other genes (denoted as beta-T1, beta-T2, beta-T3 and beta-T4). Here we report that the amino acid sequence of the carboxy-terminal domain predicted from the beta-T3 gene (apparently the most expressed of the gene family) contains six modifications (five substitutions and one insertion) of conserved residues, unique with respect to all the other known beta-tubulin sequences. These modifications can change the structural conformation of the carboxy-terminal domain. Furthermore, in the variable terminal end of that domain, a consensus sequence for a phosphorylation site is present, and the residue Glu-438, the most frequent site for polyglutamylation in beta-tubulin, is substituted by Asp. Starting from these observations, we showed that in E. focardii only alpha-tubulin is polyglutamylated, while beta-tubulin undergoes phosphorylation. Polyglutamylated microtubules appear to colocalize with cilia and microtubular bundles, all structures in which microtubules undergo a sliding process. This finding supports the idea that alpha-tubulin polyglutamylation is involved in the interaction between tubulin and motor microtubule-associated proteins. Phosphorylation, usually a rare posttranslational modification of beta-tubulin, which is found extensively distributed in the beta-tubulin of this cold-adapted organism, may play a determinant role in the dynamic of polymerization and depolymerization at low temperatures.

Chemical signaling in ciliates.

For long, our knowledge of the biology of ciliate pheromones has long relied solely upon the study of the two structurally unrelated "gamones" identified in culture filtrates of a Blepharisma species. However, the characterization of a number of polypeptide pheromones secreted by Euplotes raikovi and E. octocarinatus has now established that structural relationships of homology usually link these molecules, which is consistent with the genetic basis of the mating type systems evolved by these species. In this context, our growing appreciation of the conserved and variable elements of the pheromone architecture should foster progress in the understanding of pheromone-receptor interactions and thus, provide important clues into pheromone mechanisms of action.

Differential amplification of pheromone genes of the ciliate Euplotes raikovi.

In hypotrich ciliates, the entire silent chromosomal genome of the germinal nucleus (micronucleus) undergoes extensive DNA rearrangements that, during the development of the somatic nucleus (macronucleus) at the beginning a new cell life cycle, eventually result in the production of linear DNA molecules. These molecules represent functional genes, each one consisting of a central coding region flanked by two shorter regions, which apparently lack canonical elements for regulation of replication and transcription. These are amplified to thousands of copies in the "adult" macronucleus of the vegetative cell. We defined the extent of this amplification for allelic codominant genes which, in the macronucleus of Euplotes raikovi, encode polypeptide cell recognition factors (pheromones). This amplification was shown to be allele-specific. The copy numbers of genes coding for pheromones Er-1, Er-2, and Er-10 were determined to be 2.5-2.9 x 10(4), 0.9-1.2 x 10(4), 1.6-1.85 x 10(4) respectively, and these numbers did not appreciably vary during the vegetative cell proliferation. This differential amplification of pheromone genes was (i) independent of whether two genes coexisted in the same heterozygous cell or were separated in the corresponding homozygotes, and (ii) directly correlated with quantitative variations in mRNA synthesis and pheromone secretion. On the basis of these results, it is suggested that a mechanism of gene-specific amplification may be used by hypotrich ciliates to modulate gene expression.

Identification of the tubulin gene family and sequence determination of one beta-tubulin gene in a cold-poikilotherm protozoan, the antarctic ciliate Euplotes focardii.

Four different tubulin genes were identified in the somatic nucleus (macronucleus) of Euplotes focardii, a strictly cold-adapted, Antarctic ciliate: one of 1,800 bp for alpha-tubulin and three of 2,150, 1,900, and 1,600 bp, respectively, for beta-tubulin. Preliminarily analysed for restriction fragment length polymorphisms, these genes showed remarkable differences in organisation from tubulin genes of other ciliates which live in temperate areas and were analysed in parallel with E. focardii. The complete coding sequence of the 1,600 bp beta-tubulin gene was then determined and shown to contain unique structural features of potential importance for E. focardii microtubule organization and activity. Of eight unique substitutions detected, seven were concentrated in the large amino terminal domain of the molecule that directly interacts with the carboxy terminal region of alpha-tubulin for heterodimer formation. Sequence analysis of the cloned gene revealed, in addition, a potential new exception in the use of the genetic code by ciliates. A TAG codon was aligned in correspondence with Trp-21 which is strictly conserved in every tubulin sequence so far determined.