Improved Metal Cation Optosensing Membranes through the Incorporation of Sulphated Polysaccharides.

Optosensing chitosan-based membranes have been applied for the detection of heavy metals, especially in drinking water. The novelty of this study is based on the use of sulphated polysaccharides, in such optosensing membranes, aiming at an improved analytical performance. The sulphated polysaccharides, such as ulvan, fucoidan and chondroitin sulfate, were extracted from by-products and wastes of marine-related activities. The membranes were developed for the analysis of aluminum. The variation in the visible absorbance of the sensor membranes after the contact between the chromophore and the aluminum cation was studied. The membranes containing sulphated polysaccharides showed improved signals when compared to the chitosan-only membrane. As for the detection limits for the membranes containing ulvan, fucoidan and chondroitin sulfate, 0.17 mg L-1, 0.21 mg L-1 and 0.36 mg L-1 were obtained, respectively. The values were much lower than that obtained for the chitosan-only membrane, 0.52 mg L-1, which shows the improvement obtained from the sulphated polysaccharides. The results were obtained with the presence of CTAB in analysis solution, which forms a ternary complex with the aluminum cation and the chromophore. This resulted in an hyperchromic and batochromic shift in the absorption band. When in the presence of this surfactant, the membranes showed lower detection limits and higher selectivity.

[Oral health and precariousness in pregnant women]. / Santé bucco-dentaire et précarité chez la femme enceinte.

OBJECTIVE: Systematic full-mouth dental examination during pregnancy is an official preventive measure recently advocated by the French Health policy. The aim of this study was firstly to evaluate the oral health related to some sociodemographic factors among pregnant women, and secondly to propose this dental examination together with the routine antenatal interview. MATERIAL AND METHOD: This cross-sectional study combined several medical questionnaires with an oral examination. It concerned all pregnant women attending their routine antenatal interview in the maternity unit of the Montpellier hospital. Socioeconomic status was assessed by Epices index. So that two groups were determined : the deprived group (D), and the non-deprived group (ND). RESULTS: Oral examination revealed that 93% of the women were suffering from at least one oral disease, 74% had a periodontal disease (9% had a periodontitis), and 74% had at least one carious tooth. The mean Epices score was 30.5 and the mean number of carious teeth was significantly higher in the group D (3.4) than in the group ND (2.35), (p=0.02). The prevalence of periodontal disease or periodontitis were not significantly different between the two groups (p=0.81 and p=0.99 respectively). After stratification on the degree of dental hygiene knowledge, it was found that knowing about an adequate dental hygiene and specific preventive measures regarding pregnancy could reduce the gap between the oral health status of the two socioeconomic groups. CONCLUSION: This study showed that performing an oral examination, at the same time than the antenatal interview, could highly improve the knowledge about dental hygiene among pregnant women and the screening of oral diseases, especially for deprived population.

Development of concomitant diseases in COVID-19 critically ill patients. / Desarrollo de enfermedades concomitantes en pacientes críticos con COVID-19.

It may be necessary a consideration about the best approach to the acute concomitant problems that critical COVID-19 patients can develop. They require a rapid diagnosis and an early treatment by a multidisciplinary team. As a result, we would like to describe two clinical cases a patient with diagnosis of COVID-19 pneumonia with good respiratory evolution that, after extubation suffered an acalculous cholecystitis and a patient with COVID-19 pneumonia that suffered an overinfection with necrotising pneumonia that presented with haemoptysis and was finally treated with arterial embolisation by the interventional radiologist's team.

[Cutaneous collagenous vasculopathy: a case report and review of the literature]. / Vasculopatía colágena cutánea: aportación de un caso y revisión de la literatura.

Cutaneous collagenous vasculopathy is an idiopathic microangiopathy first described in 2000 by Salama and Rosenthal.It must not be confused with generalized essential telangiectasia. To date, all patients have been white men over the age of 50 years, most of whom had multiple pathologies, were taking multiple drugs, and had no family history of similar conditions or hemorrhagic disorders. The disease is characterized by the development of various numbers of telangiectases on the limbs, lower abdomen, chest, or back, with no involvement of the mucosas or nail bed. Histopathology shows dilated superficial cutaneous vessels with perivascular deposits of periodic acid-Schiff diastase-positive, eosinophilic hyaline material that exhibits positive immunoreactivity to collagen IV. We report a new case in a 68-year-old man with symmetrically distributed telangiectases on his forearms, lower abdomen, posterior thighs, lower legs, and dorsum of the feet.

Targeting of U2AF65 to sites of active splicing in the nucleus.

U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins.

In higher eukaryotes, the polypyrimidine-tract (Py-tract) adjacent to the 3' splice site is recognized by several proteins, including the essential splicing factor U2AF65, the splicing regulator Sex-lethal (Sxl), and polypyrimidine tract-binding protein (PTB), whose function is unknown. Iterative in vitro genetic selection was used to show that these proteins have distinct sequence preferences. The uridine-rich degenerate sequences selected by U2AF65 are similar to those present in the diverse array of natural metazoan Py-tracts. In contrast, the Sxl-consensus is a highly specific sequence, which can help explain the ability of Sxl to regulate splicing of transformer pre-mRNA and autoregulate splicing of its own pre-mRNA. The PTB-consensus is not a typical Py-tract; it can be found in certain alternatively spliced pre-mRNAs that undergo negative regulation. Here it is shown that PTB can regulate alternative splicing by selectively repressing 3' splice sites that contain a PTB-binding site.

Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected].

The mammalian splicing factor U2AF65 binds to the polypyrimidine tract adjacent to the 3' splice site and promotes assembly of U2 small nuclear ribonucleoprotein on the upstream branch point, an interaction that involves base pairing with U2 small nuclear RNA (snRNA). U2AF65 contains an RNA binding domain, required for interaction with the polypyrimidine tract, and an arginine-serine-rich (RS) region, required for U2 snRNP recruitment and splicing. Here it is reported that binding of U2AF65 to the polypyrimidine tract directed the RS domain to contact the branch point and promoted U2 snRNA-branch point base pairing even in the absence of other splicing factors. Analysis of RS domain mutants indicated that the ability of U2AF65 to contact the branch point, to promote the U2 snRNA-branch point interaction, and to support splicing are related activities, requiring only a few basic amino acids. Thus, the U2AF65 RS domain plays a direct role in modulating spliceosomal RNA-RNA interactions.

Alternative pre-mRNA splicing: the logic of combinatorial control.

Alternative splicing of mRNA precursors is a versatile mechanism of gene expression regulation that accounts for a considerable proportion of proteomic complexity in higher eukaryotes. Its modulation is achieved through the combinatorial interplay of positive and negative regulatory signals present in the RNA, which are recognized by complexes composed of members of the hnRNP and SR protein families.

The SR protein family: pleiotropic functions in pre-mRNA splicing.

A family of proteins with arginine-serine-rich domains has recently come into the limelight of studies on the mechanisms of constitutive and regulated pre-mRNA splicing. Implicated in an ever increasing variety of functions, these proteins act as driving forces during spliceosome assembly and also play decisive roles in alternative splice-site selection, suggesting that they are crucial players in the regulation of splicing during cell differentiation and development.

[Cutaneous ciliated cyst of the scrotal skin. A case report with discussion of pathogenesis]. / Quiste cutáneo ciliado en piel escrotal. Descripción de un caso con discusión patogénica de la entidad.

Cutaneous ciliated cyst (CCC) is a rare benign lesion predominantly occurring in the lower limbs of young women and exceedingly rare in males. Here, we report a case involving a previously unreported site (i.e., scrotal skin) in a 15-year-old male. We also describe pathologic and immunonohistochemical findings, review the pertinent literature and discuss their pathogenetic mechanisms. We propose that CCC could represent a morphologic pattern encompassing several pathogenetically different entities. Data we provide support the hypothesis that at least a part of CCC, specially those occurring in males, could have their origin in ciliated metaplasia of apocrine sweat glands.

Post-transcriptional regulation: the dawn of PTB.

The 'polypyrimidine-tract-binding protein' (PTB) participates in the control of alternative processing and translation of various RNAs, and may operate as a multifunctional regulator of tissue-specific gene expression.

Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65).

U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing.

Switch in 3' splice site recognition between exon definition and splicing catalysis is important for sex-lethal autoregulation.

Maintenance of female sexual identity in Drosophila melanogaster involves an autoregulatory loop in which the protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. We have used transient transfection of Drosophila Schneider cells to analyze the role of exon 3 splice sites in regulation. Our results indicate that exon 3 repression requires competition between the 5' splice sites of exons 2 and 3 but is independent of their relative strength. Two 3' splice site AG's precede exon 3. We report here that, while the distal site plays a critical role in defining the exon, the proximal site is preferentially used for the actual splicing reaction, arguing for a switch in 3' splice site recognition between exon definition and splicing catalysis. Remarkably, the presence of the two 3' splice sites is important for the efficient regulation by SXL, suggesting that SXL interferes with molecular events occurring between initial splice site communication across the exon and the splice site pairing that leads to intron removal.

Dual function for U2AF(35) in AG-dependent pre-mRNA splicing.

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.

Novel functions for 'nuclear factors' in the cytoplasm: the Sex-lethal paradigm.

In recent years, novel functions for a number of nuclear factors have been uncovered in the cytoplasm, mainly at the level of translation. These factors behave as multifunctional regulators of gene expression and many play key roles in cell differentiation and development. One of the best characterized examples is that of Sex-lethal (SXL), an RNA-binding protein that is expressed in female Drosophila flies and controls sex determination and dosage compensation. Recent findings indicate that SXL, a paradigmatic regulator of splicing, also controls translation of target mRNAs. This review attempts to summarize this evidence and provide an overview of 'nuclear factors' with roles in translation.Copyright 1998 Academic Press Limited

[Granular cell tumour of prostate diagnosed by transrectal biopsy. Which therapeutic attitude?]. / Tumor de células granulares de próstata diagnosticado en biopsia transrectal. Qué actitud tomar?

Granular cell tumour (GCT) is not an uncommon tumour of neural origin usually located on subcutaneous tissues and oral cavity. In prostate gland is exceptional, with only one case reported on the indexed literature of the last three decades. We report a case of a 63-year-old man presented with urinary complaints, enlarged prostate and increased PSA levels. The patient subsequently underwent transrectal needle biopsy which revealed GCT. The clinicpathological dilemma originated after this diagnosis is discused and the most suitable follow-up is proposed.

Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy.

The Drosophila fl(2)d gene, required for female-specific splicing of Sxl and tra pre-mRNAs, encodes a novel nuclear protein with a HQ-rich domain.

The Drosophila gene female-lethal(2)d [fl(2)d] interacts genetically with the master regulatory gene for sex determination, Sex-lethal. Both genes are required for the activation of female-specific patterns of alternative splicing on transformer and Sex-lethal pre-mRNAs. We have used P-element-mediated mutagenesis to identify the fl(2)d gene. The fl(2)d transcription unit generates two alternatively spliced mRNAs that can encode two protein isoforms differing at their amino terminus. The larger isoform contains a domain rich in histidine and glutamine but has no significant homology to proteins in databases. Several lines of evidence indicate that this protein is responsible for fl(2)d function. First, the P-element insertion that inactivates fl(2)d interrupts this ORF. Second, amino acid changes within this ORF have been identified in fl(2)d mutants, and the nature of the changes correlates with the severity of the mutations. Third, all of the phenotypes associated with fl(2)d mutations can be rescued by expression of this cDNA in transgenic flies. Fl(2)d protein can be detected in extracts from Drosophila cell lines, embryos, larvae, and adult animals, without apparent differences between sexes, as well as in adult ovaries. Consistent with a possible function in posttranscriptional regulation, Fl(2)d protein has nuclear localization and is enriched in nuclear extracts.