Authenticity of Ecuadorian Commercial Honeys

Control of honey frauds is needed in Ecuador to protect bee keepers and consumers because simple syrups and new syrups with eucalyptus are sold as genuine honeys. Authenticity of Ecuadorian commercial honeys was tested with a vortex emulsion consisting on one volume of honey:water (1:1) dilution, and two volumes of diethyl ether. This method allows a separation of phases in one minute to discriminate genuine honeys that form three phase and fake honeys that form two phases; 34 of the 42 honeys analyzed from five provinces of Ecuador were genuine. This was confirmed with 1H NMR spectra of honey dilutions in deuterated water with an enhanced amino acid region with signals for proline, phenylalanine and tyrosine. Classic quality indicators were also tested with this method (sugars, HMF), indicators of fermentation (ethanol, acetic acid), and residues of citric acid used in the syrup manufacture. One of the honeys gave a false positive for genuine, being an admixture of genuine honey with added syrup, evident for the high sucrose. Sensory analysis was the final confirmation to recognize the honey groups studied here, namely honey produced in combs by Apis mellifera, fake honey, and honey produced in cerumen pots by Geotrigona, Melipona, and Scaptotrigona. Chloroform extractions of honey were also done to search lipophilic additives in NMR spectra. This is a valuable contribution to protect honey consumers, and to develop the beekeeping industry in Ecuador

Entomological Origin of Honey Discriminated by NMR Chloroform Extracts in Ecuadorian Honey

Honeys are produced by Apis mellifera and stingless bees (Meliponini) in Ecuador. We studied honey produced in beeswax combs by Apis mellifera, and honey produced in pots by Geotrigona and Scaptotrigona bees. Chloroform extracts of honey were obtained for fast NMR spectra. The 1D spectra were acquired at 298 K, with a 600 MHz NMR Bruker instrument, using a modified double pulsed field gradient spin echoes (DPFGSE) sequence. Signals of 1H NMR spectra were integrated and used as inputs for PCA, PLS-DA analysis, and labelled sets of classes were successfully identified, enhancing the separation between the three groups of honey according to the entomological origin: A. mellifera, Geotrigona and Scaptotrigona. This procedure is therefore recommended for authenticity test of honey in Ecuador

An Intronic Polypyrimidine-rich Element Downstream of the Donor Site Modulates Cystic Fibrosis Transmembrane Conductance Regulator Exon 9 Alternative Splicing *

Two intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041–21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774–1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tract-binding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5′-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5′-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5′-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3′-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5′-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5′-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3′-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1

Splicing Factors Induce Cystic Fibrosis Transmembrane Regulator Exon 9 Skipping through a Nonevolutionary Conserved Intronic Element

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG(m) and T(n) polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG(m) and T(n) polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG(m) and T(n) polymorphism at the 3' splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibrosis

Expression of γ-globin genes in β-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin)

Introduction: β-thalassemia is caused by autosomal mutations in the β-globin gene, which induce the absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with β-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for β-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the β+/β+ and β+/β0 genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24–48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with β-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies. © The Author(s), 2022

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased fromone in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

Cis-Elements and Trans-Acting Factors Involved in CFTR Exon 9 Alternative Splicing

Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns. Several studies have identified at 3' end of intron 8 a regulatory element that is composed by a polymorphic (TG)m(T)n repeated sequence. Variations at this polymorphic locus are associated with the alternative splicing of exon 9, which results in a non-functional CFTR protein. In our previous studies, we identified TDP43 as the factor that specifically binds to the (TG)m sequence. In this thesis we demonstrate, using transient transfection experiment with a minigene system, that overexpression of TDP43 results in an increase of exon 9 skipping. This effect is more pronounced with concomitant overexpression of SF2/ ASP SR protein previously shown to inhibit CFTR exon 9 inclusion. Moreover, antisense inhibition of endogenous TDP43 expression results in increased inclusion of exon 9. The clinical and biological relevance of this finding in viva is demonstrated by our characterization of a CF patient carrying a (TG)10T9(Delta F508)/(TG)13(T)3 genotype leading to a disease-causing high proportion of exon skipping in the allele with non mutated coding sequence. We have also previously shown the presence of a splicing inhibitory element (ISS) in the intron 9. The binding of SR proteins to the ISS modulates, together with other trans-acting factors, the level of exon 9 inclusion. In this thesis we studied the region of intron 9 from the 5' splice site until the ISS. This region presents a peculiar arrangement of polypyrimidine-rich elements and we demonstrated that one of these (PY2) acts as a splicing enhancer. In fact, mutations introduced in this PY2 element -cause a decrease in exon 9 inclusion. While the factor that binds to this pyrimidine-rich enhancer element is still unknown, we provide evidence for an involvement of polypyrimidine tract binding protein (PTB) on the modulation of exon 9 inclusion. The functional effect of PTB that we report is linked to the binding of the protein to elements in CFTR intron 9. Exonic sequences are also involved in the modulation of the alternative splicing of exon 9. In fact, for the first time, we studied the effect of several natural and site-directed mutants distributed on the entire exon and we demonstrate that in some instances this mutations induce significant changes in pre-mRNA splicing, with different degree of exon skipping and inclusion. The effect of this mutations is also modulate by the length of the polymorphic (TG)m(T)n tract at 3' end of intron 9. We also identified a critical regulatory element which contains two adjacent sequences with enhancer and silencer activity. We defined this element having overlapping enhancer and silencer properties Composit Exonic Regulatory Element of Splicing (CERES). The analysis of several site directed mutants spanning the region between nucleotides 144 and 157 showed that the functional characterization of the element cannot be simply defined by the mutated position but also by the type of nucleotide substitution. All together the results reported in this thesis shed light into the regulation of the CFTR exon 9 alternative splicing. The new cis-acting elements and trans-acting factors that we have identified point to an unexpected complexity of this system that needs further investigation. In addiction, it is certain that this is not a unique situation and that exonic and intronic splicing elements widespread in all genes and currently accepted splicing models will have to be correct

NMR carbohydrate profile in tracing acacia honey authenticity

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