19 research outputs found
Incidence of chromosome numerical changes in multiple myeloma: fluorescence in situ hybridization analysis using 15 chromosome-specific probes
Fue el primer trabajo precursor de los estudios con FISH en pacientes con Mieloma múltiple continuando la exitosa línea de investigación en mieloma del grupo de Salamanca, y por añadidura, del Grupo español de mieloma. La metodología FISH es hoy en día la principal herramienta de pronóstico en mieloma múltiple en todo el mundo.[EN]The presence of complex karotypes with frequent numerical and structural abnormalities has been reported in 20 to 50% of multiple myeloma (MM) patients. This variability is mainly due to the difficulty of conventional cytogenetics to obtain tumor metaphases representative of all possible neoplastic clones in MM. To gain insight into the real incidence of numerical chromosome changes in MM we have studied by fluorescence in situ hybridization technique 15 different human chromosomes, 1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, X, and Y, in a series of 52 MM patients. In all cases, the DNA index assessed by a propidium iodide/CD38 double-staining technique with flow cytometry was simultaneously investigated for correlation, with fluorescence in situ hybridization results. Additional aims of this study were 1) to analyze whether the abnormalities detected were common to all plasma cells or were present in only a subpopulation of tumor cells, 2) to explore changes caused by disease progression, and 3) to establish possible associations among the altered chromosomes. Although the overall incidence of numerical abnormalities was 67%, this frequency increased to 80% in the 41 cases in which 7 or more chromosomes were analyzed. Trisomies were significantly more common than monosomies (84% versus 16%). Chromosomes 9 and 15 were the most frequently altered (52% and 48% of cases, respectively), with all of their abnormalities corresponding to trisomies. The most frequent losses involved chromosomes 13 (26%) and X in females (32%). Other common numerical changes corresponded to chromosomes 1 (39%), 11 (37%), 6 (32%), 3 (31%), 18 (29%), 7 (28%), and 17 (22%). By contrast, chromosomes 8(13%), 10(8%), and 12(3%) were rarely altered. DNA aneuploidy by flow cytometry was detected in 67% of patients, and a high degree of correlation was observed between the DNA index obtained by flow cytometry and the chromosome index derived from fluorescence in situ hybridization studies, calculated according to two mathematical formulas (coefficient of correlation of 0.82 and 0.91 when at least 7 or 12 chromosomes were considered, respectively). The frequency of numeric chromosome aberrations was higher in those patients with progressive disease and, interestingly, trisomy of chromosome 8 was exclusively detected in this latter group of patients. Our study shows that, with the exception of chromosome 8, a possible marker of clonal evolution, the numeric chromosome changes are present in nearly all malignant plasma cells (r > 0.84). Finally, frequent associations between chromosomal aberrations were observed (ie, chromosomes 6, 7, 9, and 17; 7 and 15; and 11 and 17). By excluding them, it was found that two triple combinations of chromosome-specific probes, chromosomes 1 and 9 together with either chromosome 13 or 15, could be a useful marker for detection of residual disease, as it permits the identification of most MM patients displaying numerical changes.University Hosptial of Salamanca
Universidad de SalamancaHospital Universitario de Salamanc
CryoEM structures of the SARS-CoV-2 spike bound to antivirals
(Póster 63)
Background: Single-particle cryoelectron microscopy (cryoEM) has played a key role in the fight against COVID-19. The
molecular mechanisms for the action of some of the currently approved drugs targeting the SARS-CoV-2 RNA-dependent
RNA polymerase, the fast developments of the current available vaccines and antibody therapies are examples of the
impact of the knowledge gained from the cryoEM structures of SARS-CoV-2 proteins in complex with proteins (ACE2 or
antibodies/nanobodies) or small compounds. Our aim is to use this technology to understand structurally how certain
antiviral compounds and proteins targeting the spike may inhibit viral entry.
Methods: 1) Production of wild-type and mutated spike and ACE2 proteins using baculovirus/insect cells. 2) Spike
binding kinetics: protein-protein and protein-small compound interactions measured by BLI Biolayer interferometry
(BLI) and/or microscale Thermophoresis (MST). 3) Buffer optimization for cryoEM grid preparation of spike variants by
thermal shift assays and negative-staining electron microscopy (NSEM). These techniques are also used to adjust the
molar ratio of spike:ACE2 and spike:small-compound complexes. 4) Structural characterization by cryoEM.
Results: At IBV-CSIC we have created a pipeline for the production and characterization of several spike variants and
ACE2 decoys. While this pipeline is described in detail in other oral/poster communications, this communication is
centered around one of the pillars within this pipeline; the structural characterization of possible drug candidates
bound to the SARS-CoV-2 spike by cryoEM. In this way, we have successfully solved structures of the spike bound to:
A) protein inhibitors as ACE2 decoys; B) a small inhibitory compound; C) mixtures of proteins and small-compound
(nanobody-heparan derivative) working cooperatively as inhibitors. These protein/drug candidates were previously
selected based on the results obtained in our interactomics platform, whereas their concentration and the buffer
conditions for cryoEM grids preparation were established based on thermal shift assays and NSEM.
Conclusion: CryoEM is a powerful tool to directly visualize the effect caused by a potential drug on a protein target. In
a short period of time we have developed this technique in our institute to be applied to the SARS-CoV-2 spike protein,
not only to obtain high-resolution structures of SARS- CoV-2 spike variants of concern (see WP4) but also to obtain the
structures of complexes of the spike with various inhibitory compounds of very different nature
Use of an interactomics pipeline to assess the potential of new antivirals against SARS-CoV-2
(Póster 80)
Background: In late 2019 SARS-CoV-2 infection appeared in China, becoming a pandemic in 2020. The scientific
community reacted rapidly, characterizing the viral genome and its encoded proteins, aiming at interfering with viral
spreading with vaccines and antivirals. The receptor binding domain (RBD) of the viral spike (S) protein plays a key role
in cell entry of the virus. It interacts with the cellular receptor for SARS-CoV-2, the membrane-bound human Angiotensin
Converting Ectoenzyme 2 (ACE2). With the goal of monitoring interference with this interaction by potential antiviral
drugs, we have set up at the Institute for Biomedicine of Valencia (IBV-CSIC) an interactomics pipeline targeting the
initial step of viral entry.
Methods: For the production part of the pipeline (pure RBD/Spike variants and soluble ACE2), see parallel poster. These
proteins allowed monitoring of the RBD/Spike-ACE2 interaction in presence or absence of potential inhibitors. Thermal
shift assays (thermofluor) were used for initial detection of compound binding at different ligand/protein ratios
and media conditions (pH, buffers, chaotropic agents). Next, binding affinity and on/off kinetics were characterized
using Biolayer interferometry (BLI), Surface plasmon resonance (SPR), Microscale Thermophoresis (MST) and/or
Isothermal titration calorimetry (ITC). For protein-protein interactions, we mostly used BLI or SPR, whereas for proteinsmall
compound analysis MST was generally best. Protein aggregation-dissociation was monitored by size exclusion
chromatography with multiangle light scattering (SEC-MALS).
Results: Candidates proven by thermal shift assays to bind to RBD/spike protein without affecting the integrity of
these proteins were subjected to quantitative affinity measurements. We successfully demonstrated that BLI, SPR and
MST can be used to follow the interactions between SARS-CoV- 2 proteins and the putative drug candidates, as well
as to monitor the interference with Spike-Ace2 binding of potential drug candidates. While BLI and SPR displayed
reproducible results in the measurement of protein-protein interaction (applied to soluble ACE2 used as a decoy),
they were less suitable for measuring the binding of small molecules. The fact that most small compounds were only
soluble in organic solvents made difficult to obtain a low signal/noise while using BLI, necessary for the assessment
of the binding. We overcame that problem by using MST. After dilution of the compounds to the final experimental
concentrations, the technique could detect a significant binding signal enough to calculate binding parameters. MST
also allowed to measure the degree of interference that each compound was having on RBD/Spike-ACE2 interaction.
The pipeline has been customized and validated with compounds of very different nature provided by different groups
belonging to the PTI and other external laboratories, as well as with different Ace2 decoys designed at the IBV.
Conclusions: The interactomics platform at the IBV has been used to successfully develop two different antiviral
approaches in order to fight COVID-19. It has allowed technical specialization of the staff as well as the development,
in a very short period of time, of two ambitious projects. We have demonstrated that we can perform interactomic
characterization for challenging projects as well as provide information about binding of antivirals to potential new
SARS-CoV-2 variants of concern
Avaliação da Casca e da Polpa Desidratada de Café (Coffea arabica L.) pela Técnica de Degradabilidade In vitro de Produção de Gás
Avaliação da casca e da polpa desidratada de café (Coffea arabica L.) pela técnica de degradabilidade In vitro de produção de gás
Com o objetivo de avaliar a casca e a polpa desidratada de café, quanto à degradabilidade in vitro pela técnica de produção de gás, conduziu-se o experimento, utilizando as cultivares de café Catuaí, Rubi e Mundo Novo. A polpa foi obtida pela despolpa úmida em despolpador mecânico e, em seguida, seca ao sol até alcançar 13% de umidade. Os materiais foram armazenados em sacos de ráfia em ambiente coberto, ventilado e seco, por um ano, amostradas em triplicata a cada 90 dias. Incubaram-se in vitro 400 mg de cada amostra (MS e FDN), em triplicata em banho maria a 39oC. A produção cumulativa de gás foi obtida nos tempos 1, 2, 3, 4, 5, 6, 12, 18, 24, 30, 36, 48, 60 e 72 horas. A cinética da produção cumulativa de gás para a MS e FDN foi analisada utilizando-se o modelo Vt = Vt1/(1 + exp(2 + 4m(L ¾ T))) e SDN pelo modelo Vt = Vt1 x (1 ¾ exp (-m x T)). A produção cumulativa de gás da fração SDN foi obtida pela diferença entre a produção cumulativa da MS e FDN. O armazenamento da casca e polpa desidratada de café melhorou a taxa de degradação e reduziu a fração fibrosa e não degradável, disponibilizando açúcares solúveis para a flora ruminal. Na casca de café de todas as cultivares, a maximização da contribuição da fração de SDN e FDN na fermentação ocorreu, respectivamente, em torno de 24 e 48 horas. A máxima produção de gás na MS da polpa ocorreu entre 48 e 60 horas, para todas as cultivares, e foi conseqüência da máxima produção de gás da fração FDN ocorrida em torno de 60 horas. Longo período de colonização pode constituir limitação no uso da casca e polpa desidratada de café, na alimentação de ruminantes, comprometendo a utilização do alimento pelos microorganismos do rúmen, devido à rápida passagem pelo rúmen.The experiment was conducted utilizing hull and dehydrated pulp of coffee cultivars Catuaí, Rubi and Mundo Novo. Pulp was obtained by moist pulping in a mechanical pulper and dried up to 13% moisture. Materials were stored in raffia bags in ventilated, moistureless environment and sun sampled every 90 days. 400 mg of each sample (DM and NDF) were incubated in vitro in triplicates in a water bath at 39ºC. The cumulative gas production was obtained at 1,2,3,4,5,5,12, 18, 24, 30, 36 48 60 and 72 hours. The kinetics of the cumulative gas output for DM and NDF as determined using the model Vt = Vt1/(1 + exp(2 + 4m(L - T))) and SDN by the model Vt = Vt1 ´ (1 - exp(-m x T)). Cumulative gas production of the SDN fraction was obtained by the difference between the cumulative production from DM and NDF. Storage of hull and dehydrated coffee pulp improved the degradation rate, reduced fibrous and undegradable fractions, releasing soluble sugars for the ruminal flora. For coffee hulls of all cultivars, the maximum fermentation contribution of SDN and NDF took place respectively around 24 and 48 hours. The maximum gas production in pulp DM occurred between 48 and 60 hours in all cultivars and was a consequence of the maximum gas production by the NDF fraction, which peaked around 60 hours. Long colonization period may be a limitation to the use of hull and dehydrated coffee pulp in ruminant feeding, impairing feed utilization by ruminal microorganisms due to the rapid passage by the rumen
Avaliação da Casca e da Polpa Desidratada de Café (Coffea arabica L.) pela Técnica de Degradabilidade In vitro de Produção de Gás
Com o objetivo de avaliar a casca e a polpa desidratada de café, quanto à degradabilidade in vitro pela técnica de produção de gás, conduziu-se o experimento, utilizando as cultivares de café Catuaí, Rubi e Mundo Novo. A polpa foi obtida pela despolpa úmida em despolpador mecânico e, em seguida, seca ao sol até alcançar 13% de umidade. Os materiais foram armazenados em sacos de ráfia em ambiente coberto, ventilado e seco, por um ano, amostradas em triplicata a cada 90 dias. Incubaram-se in vitro 400 mg de cada amostra (MS e FDN), em triplicata em banho maria a 39ºC. A produção cumulativa de gás foi obtida nos tempos 1, 2, 3, 4, 5, 6, 12, 18, 24, 30, 36, 48, 60 e 72 horas. A cinética da produção cumulativa de gás para a MS e FDN foi analisada utilizando-se o modelo Vt = Vt1/(1 + exp(2 + 4m(L -- T))) e SDN pelo modelo Vt = Vt1 x (1 -- exp (-m x T)). A produção cumulativa de gás da fração SDN foi obtida pela diferença entre a produção cumulativa da MS e FDN. O armazenamento da casca e polpa desidratada de café melhorou a taxa de degradação e reduziu a fração fibrosa e não degradável, disponibilizando açúcares solúveis para a flora ruminal. Na casca de café de todas as cultivares, a maximização da contribuição da fração de SDN e FDN na fermentação ocorreu, respectivamente, em torno de 24 e 48 horas. A máxima produção de gás na MS da polpa ocorreu entre 48 e 60 horas, para todas as cultivares, e foi conseqüência da máxima produção de gás da fração FDN ocorrida em torno de 60 horas. Longo período de colonização pode constituir limitação no uso da casca e polpa desidratada de café, na alimentação de ruminantes, comprometendo a utilização do alimento pelos microorganismos do rúmen, devido à rápida passagem pelo rúmen
Different sensitivity levels to norspermidine on biofilm formation in clinical and commensal Staphylococcus epidermidis strains
Improved synthesis and characterization of cholesteryl oleate-loaded cationic solid lipid nanoparticles with high transfection efficiency for gene therapy applications
The development of new nanoparticle formulations that are capable of high transfection efficiency without toxicity is essential to provide new tools for gene therapy. However, the issues of complex, poorly reproducible manufacturing methods, and low efficiencies during in vivo testing have prevented translation to the clinic. We have previously reported the use of cholesteryl oleate as a novel excipient for solid lipid nanoparticles (SLNs) for the development of highly efficient and nontoxic nucleic acid delivery carriers. Here, we performed an extensive characterization of this novel formulation to make the scale up under Good Manufacturing Practice (GMP) possible. We also describe the complete physicochemical and biological characterization of cholesteryl oleate-loaded SLNs to ensure the reproducibility of this formula and the preservation of its characteristics before and after the lyophilization process. We defined the best manufacturing method and studied the influence of some parameters on the obtained nanoparticles using the Quality by Design (ICH Q8) guideline to obtain cholesteryl oleate-loaded SLNs that remain stable during storage and guarantee in vitro nucleic acid delivery efficacy. Our results indicate that this improved formulation is suitable for gene therapy with the possibility of scale-up the manufacturing of nanoparticles under GMP conditions.We are grateful to many colleagues for their helpful suggestions, critical discussions, and comments. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (grant number BFU2017-89179-R) and the Andalusian Government (Excellence Project BIO-2515/2012) to C.S. and from the Spanish Ministry of Economy and Competitiveness (grant number BFU2016-79699-P) to C.H.M. Support from the European Region Development Fund (ERDF [FEDER]) is also acknowledged. M.S.-P. was supported by a fellowship from the Spanish Ministry of Education (FPU Program).Peer reviewe