Commentary on "Poor evidence for host-dependent regular RNA editing in the transcriptome of SARS-CoV-2".

Analysis of the SARS-CoV-2 transcriptome has revealed a background of low-frequency intra-host genetic changes with a strong bias towards transitions. A similar pattern is also observed when inter-host variability is considered. We and others have shown that the cellular RNA editing machinery based on ADAR and APOBEC host-deaminases could be involved in the onset of SARS-CoV-2 genetic variability. Our hypothesis is based both on similarities with other known forms of viral genome editing and on the excess of transition changes, which is difficult to explain with errors during viral replication. Zong et al. criticize our analysis on both conceptual and technical grounds. While ultimate proof of an involvement of host deaminases in viral RNA editing will depend on experimental validation, here, we address the criticism to suggest that viral RNA editing is the most reasonable explanation for the observed intra- and inter-host variability.

Nasal IL-17F is related to bronchial IL-17F/neutrophilia and exacerbations in stable atopic severe asthma.

Severe asthma (SA) is associated with neutrophil recruitment and T helper (TH )17 chemokine overexpression in bronchial biopsies. We aimed to evaluate IL-17A and IL-17F expression in nasal/bronchial lamina propria of atopic mild-to-severe asthmatics and controls in relation to neutrophilia and asthma exacerbations. Cryostat sections of nasal/bronchial biopsies obtained from 14 SA and 14 mild asthma (MA) stable atopic patients with rhinitis, and seven healthy controls were analyzed by immunohistochemistry for neutrophils, IL-17A and IL-17F expression. Atopic SA showed an increase in asthma exacerbations number, IL-17F and IL-17A expression in nasal/bronchial lamina propria compared to MA and controls, and a higher expression of bronchial neutrophils in SA compared to MA and controls. In all asthmatics, significant relationships were found between bronchial IL-17F and neutrophils/FEV1 , nasal IL-17F and bronchial neutrophil/IL-17 markers and between the latter and exacerbations, suggesting that nasal IL-17F might be informative on bronchial IL17-driven neutrophilia in atopic SA.

Nasal and lung function in competitive swimmers.

Nasal and sinusal complaints are considered common among swimmers. Aim of the present study was to evaluate the nasal and bronchial functions, before and after swimming, and the relationship between nasal resistances and FEV1 in competitive swimmers. A group of 30 competitive swimmers were examined: spirometry and nasal respiratory tests were carried out before and after swimming. Moreover, both the competitive swimmers and the 150 visitors of a swimming pool were asked to complete a specific questionnaire. In this questionnaire, 18% of the population reported nasal-sinusal symptoms after swimming. The differences between nasal volumes and resistances before and after swimming were not statistically significant. Nasal patency increased or remained unchanged in 21/30 athletes. The variations in FEV1 were not statistically significant. In conclusion, results showed that swimming is able to increase nasal patency or to leave it unchanged. Temporary worsening of the nasal patency was observed in only a few hyper-reactive patients. In the whole group, no variations, at bronchial level, were found.

Adeno-tonsillar surgery in Italy.

Indications to surgery for adeno-tonsillar inflammatory disorders and analysis of the effectiveness of surgical treatment, compared with watchful waiting strategy, continue to be the subject of scientific debate. The present investigation focuses on the surgical activity of 14 Italian Otorhinolaryngological Units between 1999 and 2004. Surgical interventions (adeno-tonsillectomy, adenoidectomy, tonsillectomy) on 26915 children (age range: 2-11 years) were considered. Data on adeno-tonsillar interventions were analysed in relation to other interventions of ENT interest, performed in the same units and in the same period. Adeno-tonsillar interventions accounted for 35.4% of all operations of ENT interest. Adeno-tonsillectomy accounted for 56.6% of overall adeno-tonsillar operations, adenoidectomy 31.6%, tonsillectomy 11.8%. The percentage for the three interventions was homogeneous in the period of the study and in the recruited units. The percentage of children who underwent adeno-tonsillar surgery in paediatric units was higher as compared to general units, as far as concerns the overall number of operations performed. In southern Italy, the number of adeno-tonsillar interventions, in general, and of adeno-tonsillectomy, in particular, was higher compared to that in northern Italy. Results of the present study suggest that environmental factors, cultural issues and local health demands, may influence indications and, therefore, the different incidence of the operations under consideration in the units taking part in the investigation.

Galectin-3: presurgical marker of thyroid follicular epithelial cell-derived carcinomas.

Preoperative follicular lesion characterisation represents an unsolved diagnostic problem in thyroid nodular disease. Although fine-needle aspiration biopsy is the most reliable preoperative diagnostic procedure, it shows inherent limitations in differentiating adenoma from follicular carcinoma and, sometimes, follicular variants of papillary carcinoma. Galectin-3 cytoplasmic neoexpression has been proposed as a peculiar feature of thyroid malignant cells, easily detectable in cytological and histological samples. The aim of this study was to re-evaluate the galectin-3 expression in a large sample of thyroid lesions using an immunohistocytochemical biotin-free detection system and a specific anti-human-galectin-3 monoclonal antibody in order to avoid the interference of technical factors, a cause of conflicting results recently reported by some authors. We analysed galectin-3 expression of 39 follicular carcinomas, 26 papillary carcinomas, and 105 adenomas in both cell-block samples and their histological counterparts. All cell-block and histological papillary carcinoma samples showed high levels of galectin-3 immunoreactivity. Thirty-four follicular carcinomas were positive, whereas 5 were negative in cell-blocks but positive in their histological counterparts. Twelve out of 105 adenomas expressed galectin-3 in cell-blocks and histological samples. The diagnostic accuracy of preoperative galectin-3 evaluation in adenomas vs follicular carcinomas was 90.0%. Galectin-3 expression was also investigated in 22 minimally-invasive follicular carcinomas. All of them showed galectin-3 immunoreactivity in both cytological and histological specimens with the exception of two cases, where galectin-3 positivity was observed only in the surgical material. The routine correct use of galectin-3, by increasing the diagnostic accuracy of conventional cytology, improves the management of thyroid nodules and can lead to a sensitive reduction of useless thyroid surgeries.

Role of fine-needle aspiration biopsy and frozen-section evaluation in the surgical management of thyroid nodules.

BACKGROUND: The role of routine frozen section (FS) in the surgical management of thyroid nodules remains uncertain. This study reviewed the role of FS in the presence of an adequate fine-needle aspiration biopsy (FNAB). METHODS: FNAB and FS were evaluated in 206 patients who had surgery for a thyroid nodule. Cytological specimens were classified as benign, malignant or suspicious. The FS diagnoses were benign, malignant or deferred. RESULTS: A cytological diagnosis was obtained in 93 nodules; the remaining 113 were classified as suspicious, of which 21 were malignant on definitive examination. The overall accuracy of FNAB was 53 per cent. FS evaluation identified 165 lesions as benign; the diagnosis was deferred until definitive histological evaluation in only eight. The overall accuracy, therefore, was 96 per cent. Routine use of FS was cost-effective; lowering the number of reoperations led to an estimated saving of about 40 per cent. CONCLUSION: These data suggest that FS remains an important tool in the surgical management of thyroid nodules and can reduce the number of patients requiring reoperation.

Mechanisms for evolving hypervariability: the case of conopeptides.

Hypervariability is a prominent feature of large gene families that mediate interactions between organisms, such as venom-derived toxins or immunoglobulins. In order to study mechanisms for evolution of hypervariability, we examined an EST-generated assemblage of 170 distinct conopeptide sequences from the venoms of five species of marine Conus snails. These sequences were assigned to eight gene families, defined by conserved elements in the signal domain and untranslated regions. Order-of-magnitude differences were observed in the expression levels of individual conopeptides, with five to seven transcripts typically comprising over 50% of the sequenced clones in a given species. The conopeptide precursor alignments revealed four striking features peculiar to the mature peptide domain: (1) an accelerated rate of nucleotide substitution, (2) a bias for transversions over transitions in nucleotide substitutions, (3) a position-specific conservation of cysteine codons within the hypervariable region, and (4) a preponderance of nonsynonymous substitutions over synonymous substitutions. We propose that the first three observations argue for a mutator mechanism targeted to mature domains in conopeptide genes, combining a protective activity specific for cysteine codons and a mutagenic polymerase that exhibits transversion bias, such as DNA polymerase V. The high D:(n)/D:(s) ratio is consistent with positive or diversifying selection, and further analyses by intraspecific/interspecific gene tree contingency tests weakly support recent diversifying selection in the evolution of conopeptides. Since only the most highly expressed transcripts segregate in gene trees according to the feeding specificity of the species, diversifying selection might be acting primarily on these sequences. The combination of a targeted mutator mechanism to generate high variability with the subsequent action of diversifying selection on highly expressed variants might explain both the hypervariability of conopeptides and the large number of unique sequences per species.

Evolving better brains: a need for neurotrophins?

The NGF family of neurotrophins has a crucial role in regulating neuron numbers during vertebrate development. Six years ago the prediction was made that invertebrates with simple nervous systems, such as Caenorhabditis elegans, would lack neurotrophins. Surprisingly, it now appears that not only C. elegans but also Drosophila melanogaster, lack homologs of the neurotrophins or their trk receptors. Furthermore, functional studies indicate that control of neuronal numbers in Drosophila is primarily dependent on steroids. By contrast, a recognizable trk homolog exists in molluscs, a phylum that includes species with the most complex nervous systems in the invertebrate kingdom. This suggests that neurotrophic signaling mechanisms might be one of the prerequisites for evolution of complex nervous systems. Expansion of the genome projects to other invertebrates, such as molluscs and coelenterates, should provide new insights on the molecular correlates of building complex brains.

[Non-auditory effects of environmental noise: a study of metallurgical and mechanical workers]. / Gli effetti extrauditivi da rumore: indagine in una popolazione di lavoratori di una industria metalmeccanica.

The present work was performed to check the non-auditory effects of environmental noise on a homogeneous worker population. The study was performed on 200 workers (factory and office workers) in a metallurgical and mechanical company: each individual underwent thorough clinical-audiometric study and filled out a multiple-choice psycho-social-attitude questionnaire. Statistical analysis of the results obtained showed a significantly higher number of non-auditory effects in subjects exposed to noise vs. those not exposed. For all workers studied, this incidence increased proportionally with age and job seniority. Moreover, it was demonstrated that there is no direct correlation between non-auditory effects and chronic traumatic hearing loss. The limited number of cases examined, however, suggests a multi-center study in order to provide better clinical-scientific and statistical-epidemiological study of this complex question involving on-the-job noise.

Indications and results of fronto-lateral laryngectomy using a combined endolaryngeal and external approach.

Patients with laryngeal anterior commissure, cord-commissure, bilateral anterior cord-commissure carcinomas (T1 and T2 N0 M0) were subjected to a new method of frontolateral laryngectomy using a combined endolaryngeal and external approach. The proposed surgical procedure allowed the removal of the anterior commissure and part of one or both vocal cords in a single unit, together with the cartilaginous framework, respecting the integrity of the superior portion of the thyroid cartilage. The internal procedure permitted an accurate delimitation of the posterior part of the laryngeal neoplasm. In particular, this was performed during suspension microlaryngoscopy using the CO(2) laser or traditional cutting tools for section of the laryngeal visceral structures to the internal surface of the thyroid cartilage. Following this, the external approach included incision of the external perichondrium along the superior edge of the thyroid cartilage and along the median line, from the incisura to the inferior edge of the thyroid cartilage. The superior opening of the larynx is made side-to-side and the epiglottis separated at the level of the superior edge of the thyroid cartilage. The ends of the section are joined together with the superior parts of the section created during the laryngeal approach. Then progressive craniocaudal detachment of the internal perichondrium is performed backwards until the endolaryngeal sections are reached and downwards to the insertion of the cord ligaments. The inferior opening of the larynx is made by a horizontal section of the cricothyroid membrane at the level of the superior edge of the cricoid cartilage. Section of the thyroid cartilage is therefore performed in a trapezoidal shape. This section involves the inferior part of the protruding corner of the thyroid cartilage. After joining the ends of the cricothyroid section with the inferior extremities of the endolaryngeal sections, the surgical specimen is removed as a single unit. The method must be used only after accurate clinical evaluation. It is mostly recommended in subjects with cord-commissure carcinomas previously treated with radiotherapy. The results obtained were extremely satisfactory both as regards survival and functional results. In all, 27/28 patients (96.4%) were free from disease. The quality of voice was satisfactory but hoarse in 30% and breathy in 70% of the patients.

Structural features of the rat GFAP gene and identification of a novel alternative transcript.

The glial fibrillary acidic protein (GFAP) is expressed in a cell-specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell-specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPalpha, beta, and gamma, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1-14; Zelenika et al. [1995] Mol. Brain Res. 30:251-258), we called this novel mRNA isoform GFAPdelta. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101-bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPdelta transcript encodes for an hypothetical GFAP where the forty-two carboxy-terminal amino acids encoded by exon VIII and IX are replaced by thirty-three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2-kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (alpha, beta, and gamma) produce hypothetical translation products differing in the amino-terminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy-terminal Tail domain.

GFAPbeta mRNA expression in the normal rat brain and after neuronal injury.

GFAPbeta mRNA is an alternative transcript of the glial fibrillary acidic protein (GFAP) gene, whose transcriptional start site is located 169 nucleotides upstream to the classical GFAPalpha mRNA. By an RT-PCR method with primers on separate exons, we were able to confirm the presence of GFAP transcripts with a longer 5' untranslated region in all the examined areas of rat brain and in primary cultures of astroglial cells. Northern blot analysis, using an oligoprobe specific for the 5' region of GFAPbeta, revealed a single hybridization band of 2.9 kb in all the brain regions examined and in primary cultures of astroglial cells. The availability of the quantitative Northern blot assay allowed further studies on the regulation of GFAPbeta expression in vivo. Since it is well-known that neuronal brain injury is one of the most powerful inducers of GFAP, we examined the expression of GFAPalpha and beta after a neurotoxic lesion in the rat hippocampus. Results obtained show a parallel increase in both GFAP transcripts with an identical time-course, suggesting that regulatory regions of the gene influence in similar way the rate of transcription at the two different start sites (alpha and beta) or that a similar post-transcriptional mechanism is involved in regulating both mRNA isoforms.

A neural-specific hypomethylated domain in the 5' flanking region of the glial fibrillary acidic protein gene.

In the present study we examined the methylation status of the glial fibrillary acidic protein (GFAP) gene promoter, analyzing various CG sites in both the human and rat gene in GFAP-expressing and nonexpressing tissues. Moreover, we studied the methylation of specific CG sites in different rat brain areas during postnatal development, in cell cultures highly enriched in specific neural- or non-neural-cell types (fibroblasts), and in human gliomas. The obtained results do not support a simple correlation between demethylation and expression of the GFAP gene but help to identify a cluster of CG sites in the 5'flanking region (from -1176 to -1471 in the rat) that are hypomethylated in neural cell types and localized in a region highly conserved between rat, mouse and human GFAP promoters. Neural-specific hypomethylation of this conserved zone can be observed also in the human GFAP gene both in normal brain tissue and neoplastic glial cells. A higher demethylation of the -1176 site at early stage of postnatal life was observed in specific rat brain areas, such as hippocampus and cerebellum. The most dramatic differences were observed in the cerebellum where a peak of demethylation of the -1176 site was detected at 15 days of postnatal life, followed by an intense remethylation of this site. Results of experiments in the CG4 glial progenitor cell line showed that demethylation of the -1176 site is already established before transcriptional activation of the GFAP gene. Moreover, results of experiments in primary cell cultures show that in neuronal cell types, such as cerebellar granule cells and embryonic cerebral hemisphere neurons, the level of demethylation of the -1176 site is comparable to that observed in cultured astrocytes. In contrast a high level of methylation can be observed in cultured non-neural cell types (fibroblasts). Such neural-specific hypomethylation could be established in a very early stage in the progression along the neural cell lineage and could play a role in maintaining a local open chromatin conformation which is then necessary to allow the interaction with specific regulatory factors present in astroglial cells.

[Fronto-lateral laryngectomy using a combined endolaryngeal external approach]. / Laringectomia fronto-laterale per via combinata, endolaringea ed esterna.

Functional laryngectomies-whether subtotal (with significant demolition) or conservative (with limited latero-frontal and frontal demolition)-can, at times, lead to significant post operative functional sequelae. Negative outcomes method proposed calls for an internal, endolaryngeal approach along with an external approach. The internal approach makes it possible to carefully delimit the posterior extension of the laryngeal neoplasm. The external approach, on the other hand, makes it possible to remove the anterior commissure and part of one or both vocal cords in a single unit together with a smaller portion of the thyroid cartilage. In particular, the endolaryngeal approach using suspension microlaryngoscopy makes it possible to section the laryngeal structures all the way up to the internal surface of the thyroid cartilage and, anteriorly, along the midline from the incision to the lower edge of the cartilage. From above the larynx is opened by means of a transfixed section of the thyroid membrane and of the epiglottis at the upper edge of the thyroid cartilage. The ends of the section are joined with the upper ends of the section prepared using the endolaryngeal approach. Cranio-caudal dissection of the internal perichondrium of the thyroid larynx is then performed, from behind, all the way to the endolaryngeal sections and, from below, to the point where the cord ligaments are inserted. The larynx is opened from below by means of a horizontal section in the crico-thyroid membrane at the upper edge of the cricoid cartilage. The trapezoidal section of the lower part of the protruding corner of the thyroid cartilage. After having joined the edges of the crico-thyroid section with the lower edge of the endolaryngeal sections, the entire piece is then removed in a single unit. This method, for treatment of carcinoma of the anterior commissure and of the anterior portion of one or both vocal cords (T1 and T2 N0 M0), must be used after accurate clinical evaluation; it is particularly appropriate for subjects with cordo-commissure carcinoma which was previously treated with radiotherapy. The method meets the requirements of oncological radicalness, permits one to perform both the external and internal sections under visual control and maintains the endolaryngeal diameters nearly intact.

Tissue-specific DNA methylation patterns of the rat glial fibrillary acidic protein gene.

The glial fibrillary acidic protein (GFAP) is an intermediate filament protein, specific of the cytoskeleton of astrocytes in the central nervous system. In the present work, as a preliminary step to the study of glial-specific gene expression, we cloned the rat GFAP gene, and we report the sequence of 1.9 kb of the 5' flanking region, exon 1, and the majority of the first intron. By digestion with methylation-sensitive restriction enzymes followed by Southern blot analysis, the methylation status of various CpG sites was examined in this genomic segment. We tested whether structural modification of the GFAP gene, such as DNA methylation, could be related to its tissue-specific transcriptional activity. Therefore, we compared a GFAP-expressing cell population (primary culture of astroglial cells), a mixed population of GFAP-expressing and -nonexpressing cells (adult rat cerebral hemispheres), and a GFAP-nonexpressing tissue (liver). In the 5' flanking region we identified a CpG site at position -1176 whose level of methylation is inversely correlated to GFAP expression. In primary cultured astrocytes, 75% of the GFAP gene alleles were demethylated at this site, while the corresponding value obtained for the cerebral hemispheres was 45%, and for liver only 9%. On the basis of the sequence data, a CpG-rich region (putative CpG island) was identified extending from -38 to +347 and overlapping 80% of the first exon. HhaI and HpaII sites located in the putative CpG island showed a relatively high level of methylation in all the cell populations examined, and did not show any clear correlation with the level of GFAP gene expression or with the methylation status of the -1176 site. Further in vivo developmental studies and in vitro differentiation studies are necessary to better understand the functional differences of the various methylatable CpG sites in the 5' end of the GFAP gene.

Pidotimod in the management of recurrent pharyngotonsillar infections in childhood.

The efficacy and safety of a new synthetic immunostimulant pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid, PGT/1A, CAS 121808-62-6) in recurrent infections of the primary airways were assessed in a group of 416 children with a history of recurrent respiratory infections (RRI). This was a double-blind randomized trial of pidotimod vs. placebo, consisting of a treatment period of 60 days and a follow-up period of 3 months. A reduction in the duration and frequency of infectious episodes in the group of children treated with pidotimod (one 400 mg oral bottle daily) was observed which was statistically different from the placebo group. The protective effect produced by pidotimod was also confirmed by a series of recordings made over the five-month observation period, which showed a significant reduction in the number of days of fever, the severity of the signs and symptoms of acute episodes, use of antibiotics and antipyretic drugs and absence from school or nursery school. Safety was excellent.

[A comparative study of the pterygopalatine fossa by computed tomography and magnetic resonance]. / Studio comparato della fossa pterigo-palatina con tomografia computerizzata e con risonanza magnetica.

The pterygopalatine fossa (PPF) is a narrow, bone lined space containing many major cranial nerves and vessels connecting it to the middle and infratemporal cranial fossa, the oral, nasal and orbital cavities and the rhinopharynx. The internal maxillary artery, the second branch of the V cranial nerve and the sphenopalatine ganglion are the main structures which are found in the PPF. Because of its strategic site, the PPF can be involved in many pathologic conditions originating from the nasal fossa, the orbit, the paranasal sinuses or the rhinopharynx roof. Severe craniofacial trauma can involve the pterygopalatine area too. Consequently, the detailed knowledge of PPF anatomy and the use of the current imaging modalities are necessary in the study of pterygopalatine lesions. We examined a group of 7 patients who underwent CT and MRI for diseases not involving the pterygopalatine area. CT consisted of axial and coronal contiguous 1-mm scans, with post-processing sagittal and 3D reconstructions. MRI was performed with a middle-field unit, using a head coil and high-resolution 3-mm T1- and T2-weighted spin- and fast-spin-echo scans. 3D SPGR pulse sequences were also performed. We found HRCT scans and post-processing 3D studies to be particularly indicated in bone structure analysis. SPGR, spin- and fast-spin-echo MR sequences allowed us to obtained reliable images of the main nervous and vascular structures found in the PPF. Finally, we believe combined CT and MRI to be the method of choice in the study of PPF anatomy and in the assessment of diseases involving the PPF area.

[The definition of a reference protocol for the clinical study of vertigo drugs]. / Definizione di un protocollo quadro per lo studio clinico di farmaci antivertiginosi.

The aim of this paper is to define the problems that arise in the clinical evaluation of drugs for the treatment of vertigo. Among these are the objective criteria used in defining vertigo and those used in evaluating efficacy of the drugs. The resulting protocol for a clinical study of vestibular drugs is a document that clarifies the debated points in the field, and above all furnishes guidelines for establishing uniformity in clinical studies. This, therefore, may become the reference protocol in Italy for clinical evaluations of drugs for the treatment of vertigo.