Application of the DNA-Specific stain methyl green in the fluorescent labeling of embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophoreconjugated phalloidi

Formylation as a Chemical Tool to Modulate the Performance of Photosensitizers Based on Boron Dipyrromethene Dimers

Heavy-atom-free photosensitizers are envisioned as the next generation of photoactive molecules for photo-theragnosis. In this approach, and after suitable irradiation, a single molecular scaffold is able to visualize and kill tumour cells by fluorescence signalling and photodynamic therapy (PDT), respectively, with minimal side effects. In this regard, BODIPY-based orthogonal dimers have irrupted as suitable candidates for this aim. Herein, we analyse the photophysical properties of a set of formyl-functionalized BODIPY dimers to ascertain their suitability as fluorescent photosensitizers. The conducted computationally aided spectroscopic study determined that the fluorescence/singlet oxygen generation dual performance of these valuable BODIPY dimers not only depends on the BODIPY-BODIPY linkage and the steric hindrance around it, but also can be modulated by proper formyl functionalization at specific chromophoric positions. Thus, we propose regioselective formylation as an effective tool to modulate such a delicate photonic balance in BODIPY-based dimeric photosensitizers. The taming of the excited-state dynamics, in particular intramolecular charge transfer as the key underlying process mediating fluorescence deactivation vs. intersystem crossing increasing, could serve to increase fluorescence for brighter bioimaging, enhance the generation of singlet oxygen for killing activity, or balance both for photo-theragnosis.This research received financial support from the Spanish Ministerio de Ciencia e Innovación (MCIN)/Agencia Estatal de Investigación (AEI) Grants: PID2020-114755GB-C32 and -C33 funded by MCIN/AEI/10.13039/501100011033. Gobierno Vasco (IT1639-22) is also grateful for the financial support

Efficient production of acetoin by fermentation using the newly isolated mutant strain Lactococcus lactis subsp. lactis CML B4

With the aim of applying biotechnology to produce acetoin, a chemical that can be used as an aroma and as a building block for other compounds, several putative mutants with reduced lactic acid synthesis were obtained from a wild-type homolactic strain of L. lactis subjected to chemical mutagenesis. Among these mutants, a strain was isolated, CML B4, that showed reduced lactate dehydrogenase (LDH) and increased NADH oxidase (NOX) activities. Shaken flask cultures of this mutant strain mainly produced acetoin, increasing the levels produced compared to the wild-type strain by 15-fold. A point mutation detected in the ldh gene encoding LDH was probably the genetic defect responsible for this phenotype. In pH-controlled aerobic batch fermentation, the CML B4 strain produced more than 40 gL−1 acetoin, which was increased by up to 59 gL−1 in fed-batch fermentations, with yields close to 88 and 74%, respectively, and productivities exceeding 2 gL−1 h−1. These results indicate that this strain could be used industrially as a cell factory for the production of acetoin from bioresources.This work was supported by the Department of Industry, Trade and Tourism of the Basque Government [grant number S-PE05LE03]

Adipose-derived stem cells combined with Neuregulin-1 delivery systems for heart tissue engineering

Myocardial infarction (MI) is the leading cause of death worldwide and extensive research has therefore been performed to find a cure. Neuregulin-1 (NRG) is a growth factor involved in cardiac repair after MI. We previously described how biocompatible and biodegradable microparticles, which are able to release NRG in a sustained manner, represent a valuable approach to avoid problems related to the short half-life after systemic administration of proteins. The effectiveness of this strategy could be improved by combining NRG with several cytokines involved in cardiac regeneration. The present study investigates the potential feasibility of using NRG-releasing particle scaffold combined with adipose derived stem cells (ADSC) as a multiple growth factor delivery-based tissue engineering strategy for implantation in the infarcted myocardium. NRG-releasing particle scaffolds with a suitable size for intramyocardial implantation were prepared by TROMS. Next, ADSC were adhered to particle scaffolds and their potential for heart administration was assessed in a MI rat model. NRG was successfully encapsulated reaching encapsulation efficiencies of 92.58 ± 3.84 %. NRG maintained its biological activity after the microencapsulation process. ADSC cells adhered efficiently to particle scaffolds within a few hours. The ADSC-cytokine delivery system developed proved to be compatible with intramyocardial administration in terms of injectability through a 23-gauge needle and tissue response. Interestingly, ADSC-scaffolds were present in the peri-infarted tissue two weeks after implantation. This proof of concept study provides important evidence required for future effectiveness studies and for the translation of this approach

Clinical relevance of the severe abnormalities of the T cell compartment in septic shock patients

8 pages, 2 figures.-- PMID: 19243622 [PubMed].[Introduction] Given the pivotal role of T lymphocytes in the immune system, patients with septic shock may show T cell abnormalities. We have characterised the T cell compartment in septic shock and assess its clinical implications.[Methods] T lymphocytes from the peripheral blood of 52 patients with septic shock and 36 healthy control subjects were analysed on admission to the intensive care unit, baseline, and 3, 7, 14 and 28 days later. T cell phenotypes (CD3+CD4+/CD3+CD8+, CD45RA+/CD45RO+, CD62L+/CD28+) were assessed by quantitative flow cytometry.[Results] CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte counts were significantly lower in patients with septic shock than control subjects. In surviving patients, CD3+CD4+ lymphocytes had normalised after 14 days, yet CD3+CD8+ numbers were still low. Non effector CD45RA+CD45RO- subsets of CD3+CD4+ and CD3+CD8+ were persistently low during patient follow up. CD3+CD8+CD28+ and CD3+CD8+CD62L+ were reduced in patients versus controls and survivors versus nonsurvivors in the first three days. A prediction receptor operative curve revealed that for the CD3+CD8+CD28+ subset, a cutoff of 136 cells/ml showed 70% sensitivity and 100% specificity for predicting death and the area under the curve was 0.84 at admission. Corresponding values for CD3+CD8+CD62L+ were 141 cells/ml, 60% sensitivity, 100% specificity and an area under the curve of 0.75.[Conclusions] A severe redistribution of T lymphocyte subsets is found in septic shock patients. A different kinetic pattern of T cell subset involvement is observed in surviving and nonsurviving patients, with lower numbers of circulating CD3+CD8+CD28+ and CD3+CD8+CD62L+ associated with a better disease outcome.This study was supported by grants S-BIO-0189/2006 MITIC/TIMEDIC from Comunidad de Madrid, Fondo de Investigaciones Sanitarias, CIBERehd and by a research prize awarded by the Fundación Lilly.Peer reviewe

Testing the Unification Model for AGN in the Infrared: are the obscuring tori of Type 1 and 2 Seyferts different?

We present new mid-infrared (MIR) imaging data for three Type-1 Seyfert galaxies obtained with T-ReCS on the Gemini-South Telescope at subarcsecond resolution. Our aim is to enlarge the sample studied in a previous work to compare the properties of Type-1 and Type-2 Seyfert tori using clumpy torus models and a Bayesian approach to fit the infrared nuclear spectral energy distributions (SEDs). Thus, the sample considered here comprises 7 Type-1, 11 Type-2, and 3 intermediate-type Seyferts. The unresolved IR emission of the Seyfert 1 galaxies can be reproduced by a combination of dust heated by the central engine and direct AGN emission, while for the Seyfert 2 nuclei only dust emission is considered. These dusty tori have physical sizes smaller than 6 pc radius, as derived from our fits. Unification schemes of AGN account for a variety of observational differences in terms of viewing geometry. However, we find evidence that strong unification may not hold, and that the immediate dusty surroundings of Type-1 and Type-2 Seyfert nuclei are intrinsically different. The Type-2 tori studied here are broader, have more clumps, and these clumps have lower optical depths than those of Type-1 tori. The larger the covering factor of the torus, the smaller the probability of having direct view of the AGN, and vice-versa. In our sample, Seyfert 2 tori have larger covering factors and smaller escape probabilities than those of Seyfert 1. All the previous differences are significant according to the Kullback-Leibler divergence. Thus, on the basis of the results presented here, the classification of a Seyfert galaxy as a Type-1 or Type-2 depends more on the intrinsic properties of the torus rather than on its mere inclination towards us, in contradiction with the simplest unification model.Comment: 21 pages, 14 figures, Appendix including supplementary figures. Accepted by Ap

High contrast optical imaging of companions: the case of the brown dwarf binary HD-130948BC

High contrast imaging at optical wavelengths is limited by the modest correction of conventional near-IR optimized AO systems.We take advantage of new fast and low-readout-noise detectors to explore the potential of fast imaging coupled to post-processing techniques to detect faint companions to stars at small separations. We have focused on I-band direct imaging of the previously detected brown dwarf binary HD130948BC,attempting to spatially resolve the L2+L2 benchmark system. We used the Lucky-Imaging instrument FastCam at the 2.5-m Nordic Telescope to obtain quasi diffraction-limited images of HD130948 with ~0.1" resolution.In order to improve the detectability of the faint binary in the vicinity of a bright (I=5.19 \pm 0.03) solar-type star,we implemented a post-processing technique based on wavelet transform filtering of the image which allows us to strongly enhance the presence of point-like sources in regions where the primary halo dominates. We detect for the first time the BD binary HD130948BC in the optical band I with a SNR~9 at 2.561"\pm 0.007" (46.5 AU) from HD130948A and confirm in two independent dataset that the object is real,as opposed to time-varying residual speckles.We do not resolve the binary, which can be explained by astrometric results posterior to our observations that predict a separation below the NOT resolution.We reach at this distance a contrast of dI = 11.30 \pm 0.11, and estimate a combined magnitude for this binary to I = 16.49 \pm 0.11 and a I-J colour 3.29 \pm 0.13. At 1", we reach a detectability 10.5 mag fainter than the primary after image post-processing. We obtain on-sky validation of a technique based on speckle imaging and wavelet-transform processing,which improves the high contrast capabilities of speckle imaging.The I-J colour measured for the BD companion is slightly bluer, but still consistent with what typically found for L2 dwarfs(~3.4-3.6).Comment: accepted in A\&

High-resolution optical imaging of the core of the globular cluster M15 with FastCam

We present high-resolution I-band imaging of the core of the globular cluster M15 obtained at the 2.5 m Nordic Optical Telescope with FastCam, a low readout noise L3CCD based instrument. Short exposure times (30 ms) were used to record 200000 images (512 x 512 pixels each) over a period of 2 hours 43 min. The lucky imaging technique was then applied to generate a final image of the cluster centre with FWHM ~ 0".1 and 13" x 13" FoV. We obtained a catalogue of objects in this region with a limiting magnitude of I=19.5. I-band photometry and astrometry are reported for 1181 stars. This is the deepest I-band observation of the M15 core at this spatial resolution. Simulations show that crowding is limiting the completeness of the catalogue. At shorter wavelengths, a similar number of objects has been reported using HST/WFPC observations of the same field. The cross-match with the available HST catalogues allowed us to produce colour-magnitude diagrams where we identify new Blue Straggler star candidates and previously known stars of this class.Comment: 11 pages, 15 figures. Accepted for publication in Monthly Notices of the Royal Astronomical Societ

The relative abundance of fecal bacterial species belonging to the Firmicutes and Bacteroidetes phyla is related to plasma levels of bile acids in young adults

The online version contains supplementary material available at https://doi.org/10.1007/s11306-023-02016-8.Funding for open access publishing: Universidad de Granada/CBUA. The study was supported by the Spanish Ministry of Economy and Competitiveness via Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III (PI13/01393) and PTA 12264-I, Retos de la Sociedad (DEP2016-79512-R), and European Regional Development Funds (ERDF), by the Spanish Ministry of Education (FPU16/05159, FPU16/02828, FPU17/01523 and FPU19/01609), the Fundacion Iberoamericana de Nutricion (FINUT), the University of Granada Plan Propio de Investigacion 2016-Excellence actions: Unit of Excellence on Exercise and Health (UCEES), AstraZeneca HealthCare Foundation, and by the Junta de Andalucia, Consejeria de Economia, Conocimiento, Empresas y Universidad (ref. P18-RT-4455), the Chinese Scholarship Council (CSC, No. 201707060012 to XD), Fundacion Alfonso Martin Escudero and Maria Zambrano fellowship by the Ministerio de Universidades y la Union Europea -NextGenerationEU (RR_C_2021_04). We would like to thank the team of Data Integration Center of University Medicine Magdeburg for local data-analysis solutions; they are supported by MIRACUM and funded by the German Federal Ministry of Education and Research (BMBF) within the "Medical Informatics Funding Scheme" (FKZ 01ZZ1801H). This study is part of a Ph.D. thesis conducted within the Biomedicine Doctoral Studies Program of the University of Granada, Spain. AL was supported by the funds from European Commission through the "European funds for regional development" (EFRE) Project ID: ZS/2018/11/95324.Background Gut bacteria play a crucial role in the metabolism of bile acids (BA). Whether an association exists between the fecal microbiota composition and circulating BA levels in humans is poorly understood. Here, we investigated the relationship between fecal microbiota diversity and composition with plasma levels of BA in young adults. Methods Fecal microbiota diversity/composition was analyzed with 16S rRNA sequencing in 80 young adults (74% women; 21.9 +/- 2.2 years old). Plasma levels of BA were measured using liquid chromatography-tandem mass spectrometry. PERMANOVA and Spearman correlation analyses were used to investigate the association between fecal microbiota parameters and plasma levels of BA. Results Fecal microbiota beta (P = 0.025) and alpha diversity indexes of evenness (rho = 0.237, P = 0.033), Shannon (rho = 0.313, P = 0.004), and inverse Simpson (rho = 0.283, P = 0.010) were positively associated with plasma levels of the secondary BA glycolithocholic acid (GLCA). The relative abundance of genera belonging to the Firmicutes and Bacteroidetes phyla was positively correlated with plasma levels of GLCA (all rho = 0.225, P = 0.049). However, the relative abundance of species from Firmicutes and Bacteroidetes phyla were negatively correlated with plasma levels of primary and secondary BA (all rho = - 0.220, P = 0.045), except for the relative abundance of Bacteroides vulgatus, Alistipes onderdonkii, and Bacteroides xylanisolvens species (Bacteroidetes phylum) that were positively correlated with the plasma levels of GLCA. Conclusions The relative abundance of specific fecal bacteria species is associated with plasma levels of BA in young adults. However, further investigations are required to validate whether the composition of the gut microbiota can regulate the plasma concentrations of BA in humans.Universidad de Granada/CBUASpanish Ministry of Economy and Competitiveness via Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III PI13/01393Retos de la Sociedad DEP2016-79512-REuropean CommissionSpanish Government FPU16/05159, FPU16/02828, FPU17/01523, FPU19/01609Fundacion Iberoamericana de Nutricion (FINUT)University of Granada Plan Propio de Investigacion 2016-Excellence actions: Unit of Excellence on Exercise and Health (UCEES)AstraZenecaJunta de Andalucia P18-RT-4455China Scholarship Council 201707060012Ministerio de Universidades y la Union Europea -Next Generation EU RR_C_2021_04MIRACUMFederal Ministry of Education & Research (BMBF) FKZ 01ZZ1801HUniversity of Granada, SpainEuropean Commission Joint Research Centre ZS/2018/11/9532