Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.     

Incorporación de la ecografía y la punción de tiroides a la actividad de endocrinología en una consulta de alta resolución

IntroductionFine-needle aspiration biopsy (FNAB) is considered the reference diagnostic procedure for thyroid nodules.Materials and methodsRoutine performance of thyroid ultrasound and ultrasound-guided FNAB by endocrinologists allows a more efficient approach in the setting of a high-resolution practice, thus reducing costs and the time elapsed until diagnosis.ResultsWe present our initial results of this procedure 2 years after its introduction, with a total of 286 biopsies performed. After a progressive learning curve over time and according to the endocrinologists’ previous experience, 72.72% samples were considered satisfactory for diagnosis. Greater difficulty was observed in obtaining optimal cytological specimens in smaller nodules. In conclusion, we have successfully incorporated thyroid ultrasound and ultrasound-guided FNAB into routine endocrine practice.ConclusionRoutine performance of thyroid ultrasound in endocrine practice will considerably aid the management of nodular thyroid disease.     

Valoración de la utilidad del test de estimulación intraarterial con calcio en el diagnóstico de localización del hiperinsulinismo endógeno

Background and objectiveThe aim of this study was to assess the utility of arterial calcium stimulation with hepatic venous sampling (ASVS) in the localization of tumors in patients with endogenous hyperinsulinism not detected with other methods.Patients and methodsWe performed a retrospective study of 26 patients admitted to our hospital for hypoglycemia who underwent ASVS because the source of hyperinsulinism was not clearly identified by other imaging techniques. The histopathological result in patients who underwent a surgical procedure was considered the reference for statistical study of the accuracy of this technique. Statistical analysis was performed by comparing proportions with the chi-squared test with Yates’ correction for contingency tables, and Cohen′s kappa coefficient as a measure of interrater agreement between two observations.ResultsSurgery was performed in 17 patients, 13 with positive ASVS and the remaining four with negative results. An insulinoma was removed in 12 patients, and 10 of these were detected in the ASVS. A total of 76.9 % of positive ASVS tests corresponded to a histological diagnosis of insulinoma, and 83% of these insulinomas were positive in ASVS. This association was statistically significant (chi cuadrado = 7.340; p = 0.012). Two of three patients with nesidioblastosis had a positive response in the ASVS. A good and statistically significant agreement was obtained between histopathologic diagnosis and ASVS results (κ=0.556, p = 0.007).ConclusionsASVS is a useful procedure in the localization diagnosis of endogenous hyperinsulinism not detected by other imaging tests. This technique allows tumors in the pancreatic gland to be identified and may be useful in the choice of the surgical technique to be used.     

Trauma and the endocrine system

The endocrine system may be the target of different types of trauma with varied consequences. The present article discusses trauma of the hypothalamic–pituitary axes, adrenal glands, gonads, and pancreas. In addition to changes in circulating hormone levels due to direct injury to these structures, there may be an endocrine response in the context of the stress caused by the trauma.     

Development,characterization, validation,and mapping of SSRs derived from <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.<Emphasis Type="Italic">–Moniliophthora perniciosa</Emphasis> interaction ESTs

In this study, we report results of the detection and analysis of SSR markers derived of cacao–Moniliophthora perniciosa expressed sequence tags (ESTs) in relation to cacao resistance to witches’ broom disease (WBD), and we compare the polymorphism of those ESTs (EST-simple sequence repeat (SSR)) with classical neutral SSR markers. A total of 3,487 ESTs was used in this investigation. SSRs were identified in 430 sequences: 277 from the resistant genotype TSH 1188 and 153 from the susceptible one Catongo, totalizing 505 EST-SSRs with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%), and tetranucleotides (0.6%). EST-SSRs were classified into 16 main categories; most of the EST-SSRs belonged to “Unknown function” and “No homology” categories (45.82%). A high frequency of SSRs was found in the 5’UTR and in the ORF (about 27%) and a low frequency was observed in the 3’UTR (about 8%). Forty-nine EST-SSR primers were designed and evaluated in 21 cacao accessions, 12 revealed polymorphism, having 47 alleles in total, with an average of 3.92 alleles per locus. On the other hand, the 11 genomic SSR markers revealed a total of 47 alleles, with an average of 5.22 alleles per locus. The association of EST-SSR with the genomic SSR enhanced the analysis of genetic distance among the genotypes. Among the 12 polymorphic EST-SSR markers, two were mapped on the F₂ Sca 6 × ICS 1 population reference for WBD resistance.     

A new species of Phyllotis (Rodentia,Cricetidae, Sigmodontinae) from Tucumán province,Argentina

A new species of rodent of the genus Phyllotis is described based in cranial and external morphology, as well as morphometric data. Additionally, sequences of the mitochondrial gene cytochrome-b were used to asses the phylogenetic relationships. We have compared our specimens with all the extant species of the genus Phyllotis and also with some species of related genera, particularly with the most similar and with those that occur in the province of Tucumán and northwestern Argentina. The new species is large compared to the average size of the genus, and can be easily distinguished from all other species essentially by coloration and by cranial morphology. It is closely related to the recently described P. anitae, and these two species are, in turn, sister to P. osilae. The only two localities where the new species has been found are in the Upper Montane Forests of the southern portion of the Yungas Ecoregion, in the province of Tucumán, Argentina.     

A review of research on Chinese <Emphasis Type="Italic">Tuber</Emphasis> species

Truffles are abundant in some regions of China. Nevertheless, it was not until the 1980s that Tuber species were discovered by Chinese mycologists. In recent years, international truffle markets have shown an increasing interest in the import of Chinese Tuber. These truffles serve as a complement to European truffles due to their lower prices and their greater availability in a deficit market. However, Chinese truffles have been the subject of fraudulent commercial practices, and these could have a negative effect on truffle culture. These concerns have been behind numerous recent studies designed to obtain detailed information about Chinese Tuber species. Unfortunately, many of these studies are not published in English, and are dispersed throughout the specific local or national bibliography and proceedings of specialized truffle conferences. In view of the need to expand current knowledge of Chinese Tuber species, we present a comprehensive summary of the taxonomy, ecology, mycorrhizae, genetics, biochemistry, and cultivation of Chinese Tuber species. We also provide a synthetic taxonomy and morphological characterization of 16 Chinese Tuber species in order to assist in their verification and monitoring.     

The costal skeleton of Homo antecessor: preliminary results

The Lower Pleistocene TD6 level at the Gran Dolina site in the Sierra de Atapuerca (Burgos, Spain) has yielded nine ribs that represent a minimum of three individuals of the species, Homo antecessor. We present a detailed morphological and metric study of these costal elements, including the siding and anatomical position of all of the rib remains. The adult or nearly adult ribs are also metrically compared with other fossil hominins and with modern comparative samples. The costal elements recovered to date from the TD6 level at Gran Dolina can neither confirm nor reject the hypothesis that H. antecessor had a large thorax, similar to that of Neandertals. However, the fragmentary evidence of the H. antecessor thoracic skeleton is not inconsistent with this suggestion based on other skeletal elements, such as clavicles.

Resumen

En el nivel TD6 del Pleistoceno inferior del yacimiento de Gran Dolina, en la Sierra de Atapuerca (Burgos, España) se han recuperado nueve costillas que pertenecen a un mínimo de tres individuos de la especie Homo antecessor. Presentamos un detallado estudio métrico y morfológico incluyendo el lado y la determinación anatómica. Las costillas pertenecientes a individuos adultos o casi adultos también son comparadas métricamente a muestras modernas de comparación y otros homininos fósiles. Basándonos en el registro de costillas de Homo antecessor recuperado hasta el momento no podemos probar ni refutar la hipótesis de que esta especie presentaba un tórax grande similar al de los Neandertales. Sin embargo, el registro de costillas no es inconsistente con la hipótesis de un tórax grande como sugiere la gran longitud de sus clavículas.     

Association of Hemolytic Activity of Pseudomonas entomophila,a Versatile Soil Bacterium,with Cyclic Lipopeptide Production

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C₁₀OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.Pseudomonas entomophila is a recently isolated Pseudomonas species that is closely related to the saprophytic soil bacterium Pseudomonas putida. It was initially characterized as a natural pathogen of Drosophila (63). Indeed, P. entomophila was first isolated from flies sampled in Guadeloupe, and it is highly pathogenic for Drosophila larvae and adults. P. entomophila can also effectively kill members of other insect orders (e.g., Bombyx mori, Anopheles gambiae), which makes it a new entomopathogenic bacterium. Its ability to infect and kill Drosophila melanogaster very efficiently after ingestion makes it an appropriate model for the study of host-pathogen interactions (38, 62, 63).In order to unravel features contributing to the entomopathogenic properties of P. entomophila, its genome was sequenced. The results suggest that this strain is a ubiquitous, metabolically versatile bacterium that may colonize diverse habitats, including soil, rhizosphere, and aquatic systems, as shown for P. putida KT2440 (62). However, in contrast to the P. putida genome, the P. entomophila genome contains many genes that are predicted to be important for virulence toward insects. Notably, P. entomophila could secrete many degradative enzymes (proteases and lipases), putative toxins, and secondary metabolites (62). Similar factors have been shown to play a key role in the virulence of other entomopathogenic bacteria like Photorhabdus and Xenorhabdus sp. (27, 29).Insertional mutagenesis allowed the identification of several P. entomophila genes required to infect and/or kill Drosophila. This analysis demonstrated that P. entomophila virulence is under the control of the GacS/GacA two-component system (62, 63), a global regulatory system which is known to control secondary metabolite production, protein secretion, and pathogenic abilities in gammaproteobacteria (37, 65). Another study indicates that P. entomophila can counteract the Drosophila gut immune response as a result of the secretion of an abundant protease, AprA, which degrades antimicrobial peptides produced by gut epithelia and thereby promotes bacterial persistence (38). However, an AprA-deficient mutant remains virulent to some extent, indicating that P. entomophila virulence is multifactorial, AprA being one virulence factor among others.The secretion of virulence factors is a common mechanism employed by pathogens to compromise host defenses. Several entomopathogenic bacteria (e.g., Photorhabdus luminescens) secrete toxins that allow them to impair host function (8). The starting point of this study was the observation that, in contrast to several other Pseudomonas strains, P. entomophila secretes a strong diffusible hemolytic activity (which is also controlled by the Gac system). This raises the possibility of a link between this hemolytic activity and the pathogenicity of P. entomophila for Drosophila. Indeed, bacterial hemolysins are exotoxins that attack blood cell membranes and cause cell rupture by poorly defined mechanisms. It was conceivable that this hemolytic activity could be a readout for the ability of P. entomophila to damage the epithelial cells of the Drosophila gut, which plays a crucial role in its virulence (10, 33, 63).In this study, the P. entomophila hemolytic factor was identified as a cyclic lipopeptide (CLP) whose structure was elucidated. CLPs are versatile molecules with antimicrobial, cytotoxic, and surfactant properties that are produced by members of the genera Bacillus, Serratia, Burkholderia, and Pseudomonas (31, 41, 43, 50). They are produced by a ribosome-independent mechanism that utilizes multifunctional enzymes called nonribosomal peptide synthetases (NRPSs) (42, 59). These NRPSs are composed of repeated amino acid activation modules containing domains for condensation, aminoacyl adenylation, and thiolation. Modules are responsible for activation and incorporation of amino acids into the growing peptide. A large number of prokaryotic and some eukaryotic organisms synthesize peptide metabolites via this nonribosomal mechanism of biosynthesis (42, 47).Several genes involved in P. entomophila lipopeptide production were identified, three of them encoding NRPSs. The physiological role of this lipopeptide was also investigated, and it does not seem to play a role in the process of virulence towards Drosophila and Dictyostelium or in the P. entomophila biocontrol activity that was uncovered by this study. This suggests that the lifestyle of this newly identified bacterium is probably quite versatile and that lipopeptide production could be required only under specific circumstances.