In vivo mutagenesis systems accelerate directed protein evolution but often show restricted capabilities and deleterious off-site mutations on cells. To overcome these limitations, here we report an ...in vivo platform to diversify specific DNA segments based on protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) that recognizes a cognate promoter oriented towards the target sequence. Transcriptional elongation of these fusions generates transitions C to T or A to G on both DNA strands and in long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead Cas9 (dCas9) is tethered with custom-designed crRNAs as a "roadblock" for BD-T7RNAP elongation. Using this T7-targeted dCas9-limited in vivo mutagenesis (T7-DIVA) system, rapid molecular evolution of the antibiotic resistance gene TEM-1 is achieved. While the efficiency is demonstrated in E. coli, the system can be adapted to a variety of bacterial and eukaryotic hosts.
The fission yeast Schizosaccharomyces pombe is an excellent model system in which to study the coordination of cell growth and cell differentiation. In the presence of nutrients, fission yeast cells ...grow and divide; in the absence of nutrients, they stop growing and undergo cell differentiation. The molecular mechanisms underlying this response are not fully understood. Here, we demonstrate that Tor2, a fission yeast member of the TOR protein kinase family, is central to controlling the switch between cell growth and cell differentiation in response to nutrient availability. Tor2 controls cell growth and ribosome biogenesis by regulating ribosomal protein gene expression. We have found that Tor2 has an additional function in repressing sexual differentiation. Tor2 overexpression strongly represses mating, meiosis and sporulation efficiency, whereas Tor2 inactivation has the opposite effect, leading to cell differentiation, regardless of the nutritional conditions. This newly revealed function of Tor2 appears to operate by interfering with the functions of the transcription factor Ste11 and the meiosis-promoting RNA-binding protein Mei2. Thus, our data reveal a unique regulatory function of the Tor pathway - ensuring that growth and cell differentiation become mutually exclusive and that the choice between them depends on environmental conditions.
La actividad diplomática del secretario Juan de Necolalde en Londres durante los años 1631-1637 nos legó un impresionante documento en forma de las cuentas de la embajada de más de doscientos ...folios. El objetivo de este artículo es dar a conocer dicha fuente y sus posibilidades de explotación para reconstruir las actividades diplomáticas del secretario, la distribución de los gastos y las redes de sociabilidad y de diplomacia informal en torno a la embajada española. Asimismo, este análisis demuestra los relativamente amplios márgenes de actuación de un secretario de embajada, su flexibilidad a la hora de manejar la diplomacia informal y recurrir a redes de información alternativas y la integración de las esferas mercantiles, diplomáticas, religiosas y públicas en torno a la figura del secretario diplomático.
The information provided by SARS‐CoV‐2 spike (S)‐targeting immunoassays can be instrumental in clinical‐decision making. We compared the performance of the Elecsys® Anti‐SARS‐CoV‐2 S assay (Roche ...Diagnostics) and the LIAISON® SARS‐CoV‐2 TrimericS IgG assay (DiaSorin) using a total of 1176 sera from 797 individuals, of which 286 were from vaccinated‐SARS‐CoV‐2/experienced (Vac‐Ex), 581 from vaccinated/naïve (Vac‐N), 147 from unvaccinated/experienced (Unvac‐Ex), and 162 from unvaccinated/naïve (Unvac‐N) individuals. The Roche assay returned a higher number of positive results (907 vs. 790; p = 0.45; overall sensitivity: 89.3% vs. 77.6%). The concordance between results provided by the two immunoassays was higher for sera from Vac‐N (ϰ: 0.58; interquartile ranges IQR: 0.50−0.65) than for sera from Vac‐Ex (ϰ: 0.19; IQR: −0.14 to 0.52) or Unvac‐Ex (ϰ: 0.18; IQR: 0.06−0.30). Discordant results occurred more frequently among sera from Unvac‐Ex (34.7%) followed by Vac‐N (14.6%) and Vac‐Ex (2.7%). Antibody levels quantified by both immunoassays were not significantly different when <250 (p = 0.87) or <1000 BAU/ml (p = 0.13); in contrast, for sera ≥1000 BAU/ml, the Roche assay returned significantly higher values than the DiaSorin assay (p < 0.008). Neutralizing antibody titers (NtAb) were measured in 127 sera from Vac‐Ex or Vac‐N using a S‐pseudotyped virus neutralization assay of Wuhan‐Hu‐1, Omicron BA.1, and Omicron BA.2. The correlation between antibody levels and NtAb titers was higher for sera from Vac‐N than those from Vac‐Ex, irrespective of the (sub)variant considered. In conclusion, neither qualitative nor quantitative results returned by both immunoassays are interchangeable. The performance of both assays was found to be greatly influenced by the vaccination and SARS‐CoV‐2 infection status of individuals.
The effect of repetitive training on learned behavior has been an important subject in neuroscience. In instrumental conditioning in mammals, learned action early in training is often goal-driven and ...controlled by outcome expectancy, but as training progresses, it becomes more habitual and insensitive to outcome devaluation. Similarly, we recently showed in Pavlovian conditioning in crickets ("Gryllus bimaculatus") that a conditioned response (CR) is initially sensitive to devaluation of the unconditioned stimulus but becomes insensitive to it after extended training. It is known that habitual responses after extended instrumental training are characterized by a higher context specificity than are initial goal-directed actions in mammals. In this study, we investigated whether this is applicable to Pavlovian conditioning in crickets. In crickets that received a standard amount of training to associate an odor with water reward under illumination, CR under illumination was stronger than that in the dark. In crickets that received extended training under illumination, on the other hand, the level of CR did not differ in different light conditions. Further experiments confirmed that context specificity decreases with the development of behavioral automaticity by extended training, as opposed to findings in instrumental training in mammals. We conclude that the nature of habitual behaviors after extended training differs in different learning systems of animals.
In associative learning in mammals, it is widely accepted that learning is determined by the prediction error, i.e., the error between the actual reward and the reward predicted by the animal. ...However, it is unclear whether error-based learning theories are applicable to the learning occurring in other non-mammalian species. Here, we examined whether overexpectation, a phenomenon that supports error-based learning theories, occurs in crickets. Crickets were independently trained with two different conditioned stimuli (CSs), an odour and a visual pattern, that were followed by an appetitive unconditioned stimulus (US). Then the two CSs were presented simultaneously as a compound, followed by the same US. This treatment resulted in a reduced conditioned response to the odour CS when tested immediately after training. However, the response to the CS was partially recovered after 1 day. These results are the first to show overexpectation and its spontaneous recovery in an invertebrate species. While the results showing overexpectation are in agreement with the prediction by the Rescorla-Wagner model, a major form of error-based learning theories, the ones showing spontaneous recovery are not. Our results suggest that conventional error-based learning models account for some, but not for all essential features of Pavlovian conditioning in crickets.
Desmin (
) mutations cause severe skeletal and cardiac muscle disease with heterogeneous phenotypes. Recently,
mutations were described in patients with inherited arrhythmogenic right ventricular ...cardiomyopathy/dysplasia, although their cellular and molecular pathomechanisms are not precisely known. Our aim is to describe clinically and functionally the novel
-p.Glu401Asp mutation as a cause of inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia.
We identified the novel
mutation p.Glu401Asp in a large Spanish family with inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia and a high incidence of adverse cardiac events. A full clinical evaluation was performed on all mutation carriers and noncarriers to establish clinical and genetic cosegregation. In addition, desmin, and intercalar disc-related proteins expression were histologically analyzed in explanted cardiac tissue affected by the
mutation. Furthermore, mesenchymal stem cells were isolated and cultured from 2 family members with the
mutation (1 with mild and 1 with severe symptomatology) and a member without the mutation (control) and differentiated ex vivo to cardiomyocytes. Then, important genes related to cardiac differentiation and function were analyzed by real-time quantitative polymerase chain reaction. Finally, the p.Glu401Asp mutated
gene was transfected into cell lines and analyzed by confocal microscopy.
Of the 66 family members screened for the
-p.Glu401Asp mutation, 23 of them were positive, 6 were obligate carriers, and 2 were likely carriers. One hundred percent of genotype-positive patients presented data consistent with inherited arrhythmogenic cardiomyopathy/dysplasia phenotype with variable disease severity expression, high-incidence of sudden cardiac death, and absence of skeletal myopathy or conduction system disorders. Immunohistochemistry was compatible with inherited arrhythmogenic cardiomyopathy/dysplasia, and the functional study showed an abnormal growth pattern and cellular adhesion, reduced desmin RNA expression, and some other membrane proteins, as well, and desmin aggregates in transfected cells expressing the mutant desmin.
The
-p.Glu401Asp mutation causes predominant inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia with a high incidence of adverse clinical events in the absence of skeletal myopathy or conduction system disorders. The pathogenic mechanism probably corresponds to an alteration in desmin dimer and oligomer assembly and its connection with membrane proteins within the intercalated disc.
Supervised machine learning (ML) methods have been used to predict antibody responses elicited by COVID‐19 vaccines in a variety of clinical settings. Here, we explored the reliability of a ML ...approach to predict the presence of detectable neutralizing antibody responses (NtAb) against Omicron BA.2 and BA.4/5 sublineages in the general population. Anti‐SARS‐CoV‐2 receptor‐binding domain (RBD) total antibodies were measured by the Elecsys® Anti‐SARS‐CoV‐2 S assay (Roche Diagnostics) in all participants. NtAbs against Omicron BA.2 and BA4/5 were measured using a SARS‐CoV‐2 S pseudotyped neutralization assay in 100 randomly selected sera. A ML model was built using the variables of age, vaccination (number of doses) and SARS‐CoV‐2 infection status. The model was trained in a cohort (TC) comprising 931 participants and validated in an external cohort (VC) including 787 individuals. Receiver operating characteristics analysis indicated that an anti‐SARS‐CoV‐2 RBD total antibody threshold of 2300 BAU/mL best discriminated between participants either exhibiting or not detectable Omicron BA.2 and Omicron BA.4/5‐Spike targeted NtAb responses (87% and 84% precision, respectively). The ML model correctly classified 88% (793/901) of participants in the TC: 717/749 (95.7%) of those displaying ≥2300 BAU/mL and 76/152 (50%) of those exhibiting antibody levels <2300 BAU/mL. The model performed better in vaccinated participants, either with or without prior SARS‐CoV‐2 infection. The overall accuracy of the ML model in the VC was comparable. Our ML model, based upon a few easily collected parameters for predicting neutralizing activity against Omicron BA.2 and BA.4/5 (sub)variants circumvents the need to perform not only neutralization assays, but also anti‐S serological tests, thus potentially saving costs in the setting of large seroprevalence studies.
During the years 1678-1684 the Southern region of Andalusia suffered
the ravages of the plague, an earthquake, drought and floods. The aim of this article is to analyse the oral and printed ...communicative strategies developed by the
Catholic Church during this period in order to analyse how the ecclesiastical hierarchies shaped a specific account of the events, resorted to various communicative
resources and actively participated in the creation of a collective memory
n. Durante los años 1678-1684 la región sur de Andalucía sufrió los
estragos de la peste, un terremoto, la sequía y las inundaciones. El objetivo de este
artículo es analizar las estrategias comunicativas orales e impresas desarrolladas por la Iglesia católica durante este período para analizar cómo las jerarquías
eclesiásticas conformaron un relato singular de los hechos, acudieron a diversos
recursos comunicativos y participaron de manera activa en la creación de una
memoria colectiva
Recent advances in uveal melanoma treatment Álvarez‐Rodríguez, Beatriz; Latorre, Alfonso; Posch, Christian ...
Medicinal research reviews,
November 2017, Letnik:
37, Številka:
6
Journal Article
Recenzirano
Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Recent advances in the understanding of molecular characteristics helped to determine which tumors are most likely to ...progress. About 50% of patients carrying genetic alterations such as chromosomal aberrations and mutations are at significant risk for metastatic disease of which the majority will succumb to UM within few months. Currently, there is no effective treatment for metastatic uveal melanoma, and we hope this review will encourage researchers and clinicians to work to find a better standard of care. In this article we provide a comprehensive overview of the molecular framework of UM, highlighting the most common mutations involved in this kind of cancer. It also covers the most recent treatments from basic research to clinical trials, including small molecules, nucleic acids or immunotherapy, among others. It is intended to serve as a key reference for clinicians and researchers working in this field.
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