EFFECTIVENESS OF BLOCK ITERATIVE SCHEMES IN COMPUTING THE SEISMIC RESPONSE OF BUILDINGS WITH NONLINEAR BASE ISOLATION

The conventional form of solving the equations of motion governing the seismic response of building structures with nonlinear base isolation consists of using monolithic step by step integrationmethods. As an alternative, the application of a block iterative scheme is examined in this paper. After describing the particularities of the equations of motion of the structure with base isolation, different block iterative schemes are described. Their effectiveness is discussed and compared with that of monolithic step by step integration methods. Adequate examples are used with the aim of performing numerical comparisons

Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions, while retaining their specific mode of action. Gaining a deeper understanding of the drivers of instability across different stress conditions, will potentially enable the engineering of protein scaffolds that are inherently manufacturable and stable. Here, we compared the structural robustness of a humanized antibody fragment (Fab) A33 using atomistic molecular dynamics simulations under two different stresses of low pH and high temperature. RMSD calculations, structural alignments and contact analysis revealed that low pH unfolding was initiated through loss of contacts at the constant domain interface (CL-CH1), prior to CL domain unfolding. By contrast, thermal unfolding began with loss of contacts in both the CL-CH1 and variable domain interface (VL-VH), followed by domain unfolding of CL and also of VH, thus revealing divergent unfolding pathways. FoldX and Rosetta both agreed that mutations at the CL-CH1 interface have the greatest potential for increasing the stability of Fab A33. Additionally, packing density calculations found these residues to be under-packed relative to other inter-domain residues. Two salt bridges were identified that possibly drive the conformational change at low pH, while at high temperature, salt bridges were lost and reformed quickly, and not always with the same partner, thus contributing to an overall destabilization. Sequence entropy analysis of existing Fab sequences revealed considerable scope for further engineering, where certain natural mutations agreed with FoldX and Rosetta predictions. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR), which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, which provide interesting targets for future protein engineering work aimed at stabilizing to both thermal and pH-stresses simultaneously

Comparison of the pH- and thermally-induced fluctuations of a therapeutic antibody Fab fragment by molecular dynamics simulation

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions. A deeper understanding of the drivers of instability across different stress conditions, will enable the engineering of more robust protein scaffolds. We compared the impacts of low pH and high temperature stresses on the structure of a humanized antibody fragment (Fab) A33, using atomistic molecular dynamics simulations, using a recent 2.5 Å crystal structure. This revealed that low-pH induced the loss of native contacts in the domain CL. By contrast, thermal stress led to 5–7% loss of native contacts in all four domains, and simultaneous loss of >30% of native contacts in the VL-VH and CL-CH interfaces. This revealed divergent destabilising pathways under the two different stresses. The underlying cause of instability was probed using FoldX and Rosetta mutation analysis, and packing density calculations. These agreed that mutations in the CL domain, and CL-CH1 interface have the greatest potential for stabilisation of Fab A33. Several key salt bridge losses underpinned the conformational change in CL at low pH, whereas at high temperature, salt bridges became more dynamic, thus contributing to an overall destabilization. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR) to solvent, which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, and the VH and CL domains as interesting future targets for engineering stability to both pH- and thermal-stresses simultaneously

Piezoelectric coupling as a feature sensitive to structural alterations

The paper presents an exploratory study about the use of electro-mechanical coupling measurements as a feature for detecting structural alterations using piezoelectric transducers. This coupling between the piezoelectric transducers and the structure can be estimated by means of low frequency vibration measurements and will prove to be a feature sensitive to structure alterations and scarcely influenced by temperature variations. Moreover, the approach proposed is designed such that it can also employ piezoelectric elements already available in the system for other purposes (e.g., vibration control), thus not requiring any additional expensive device, and can work while the system to be monitored is operating. The paper introduces at first the principle of the method and shows how to improve its accuracy by connecting the piezoelectric transducers with proper electric impedances. Then, numerical simulations and experimental results are presented to prove the feasibility of the proposed approach

“Ellas a estudiar y bailar, ellos a hacer deporte”: Un estudio de las actividades extraescolares de los adolescentes mediante los presupuestos de tiempo

Los estudios sobre actividades extraescolares en la adolescencia muestran que el tiempo no escolar puede utilizarse para mejorar el rendimiento académico y el desarrollo de la persona, pero también es posible emplearlo en conductas de riesgo o adicciones. Estos matices diferenciales pueden analizarse mediante un presupuesto de tiempo (PT), que además de registrar las actividades practicadas, incorpora la diversidad de las mismasdentro del repertorio del adolescente, la distribución y la cantidad de tiempo destinado en distintos días y las personas que les acompañan en cada práctica. Con base en esta sensibilidad, nuestro objetivo fue conocer los usos del tiempo al salir de la escuela, atendiendo a las relaciones interpersonales y los patrones de repetición en las actividades extraescolares. Participaron 103 adolescentes (43 chicas, 60 chicos), entre 11 y 18 años (M = 14.1; DT = 2.0). Se utilizó un PT ad hoc, registrándose durante una semana las actividades —físico-deportivas, de formación o artísticas—, realizadas fuera del horario escolar, especificando con quién se realizaba cada actividad. Los resultados obtenidos muestran escasa multiactividad, diferencias entre chicas y chicos respecto a la realización de actividades, momentos y tiempos de prácticas; ciertos patrones de frecuencia temporal de práctica; diferencias en términos de horas de dedicación según actividad o género, y predominio de la presencia de los pares. Los datos recogidos muestranla utilidad de los PT para orientar estrategias de intervención dirigidas a potenciar o mejorar las jornadas extraescolares, y así incidir positivamente sobre los tiempos escolares y no escolares

Evaluation of Continuous UVC Treatments and its Combination with UHPH on Spores of Bacillus subtilis in Whole and Skim Milk

The aim of this study was to evaluate the effectiveness of different UVC treatments, alone or in combination with ultra-high pressure homogenization (UHPH) on Bacillus subtilis spores in milk. Spores of B. subtilis (CECT4002) were inoculated in whole and skim milk to an initial concentration about 6 log CFU/mL. Milk was subjected to different ultraviolet radiation treatments at 254 nm (UVC) using a concentric tubular reactor in a dose ranging from 10 to 160 J/mL. Different number of passes were used to adjust the final dose received by the matrix. In general, increasing the number of passes (defined as number of entries to the tunnel-NET) increased the inactivation of spores of B. subtilis. The best lethality results (above 4 Log CFU/mL) were obtained by applying doses from 100 J/mL with several NET. When the same doses were achieved with a single pass lethality in most cases did not exceed 1 log CFU/mL. Increasing the NET also increased the likelihood for the spores to remain longer in the effective distance from the UVC source, estimated as 0.02 mm for whole milk and 0.06 mm for skim milk. Combination of UHPH and UVC did not clearly increase the efficiency of a single UVC treatment, and a lower lethality was even observed in some cases. UHPH treatments increased the turbidity and absorption coefficient (254 nm) of both whole and skim milk

Drastic Microbial Count Reduction in Soy Milk Using Continuous Short-Wave Ultraviolet Treatments in a Tubular Annular Thin Film UV-C Reactor

Vegetative cells of Listeria monocytogenes and Escherichia coli and spores of Bacillus subtilis and Aspergillus niger were inoculated in soy milk at an initial concentration of ≈5 log CFU/mL. Inoculated and control (non-inoculated) soy milk samples were submitted to three types of treatments using a tubular annular thin film short-wave ultraviolet (UV-C) reactor with 1 mm of layer thickness. Treatments applied depended on the flow rate and the number of entries to the reactor, with UV-C doses ranging from 20 to 160 J/mL. The number of entries into the reactor tube (NET) was established as the most determining parameter for the efficiency of the UV-C treatments. Conidiospores of A. niger were reported as the most resistant, followed by B. subtilis spores, while vegetative cells were the most sensible to UV-C, with Listeria monocytogenes being more sensible than Escherichia coli. Treatments of just 80 J/mL were needed to achieve a 5 log CFU/mL reduction of L. monocytogenes while 160 J/mL was necessary to achieve a similar reduction for A. niger spores

Developmental RNA-Seq transcriptomics of haploid germ cells and spermatozoa uncovers novel pathways associated with teleost spermiogenesis

In non-mammalian vertebrates, the molecular mechanisms involved in the transformation of haploid germ cells (HGCs) into spermatozoa (spermiogenesis) are largely unknown. Here, we investigated this process in the marine teleost gilthead seabream (Sparus aurata) through the examination of the changes in the transcriptome between cell-sorted HGCs and ejaculated sperm (SPZ). Samples were collected under strict quality controls employing immunofluorescence microscopy as well as by determining the sperm motion kinematic parameters by computer-assisted sperm analysis. Deep sequencing by RNA-seq identified a total of 7286 differentially expressed genes (DEGs) (p-value < 0.01) between both cell types, of which nearly half were upregulated in SPZ compared to HCGs. In addition, approximately 9000 long non-coding RNAs (lncRNAs) were found, of which 56% were accumulated or emerged de novo in SPZ. The upregulated transcripts are involved in transcriptional and translational regulation, chromatin and cytoskeleton organization, metabolic processes such as glycolysis and oxidative phosphorylation, and also include a number of ion and water channels, exchangers, transporters and receptors. Pathway analysis conducted on DEGs identified 37 different signaling pathways enriched in SPZ, including 13 receptor pathways, from which the most predominant correspond to the chemokine and cytokine, gonadotropin-releasing hormone receptor and platelet derived growth factor signaling pathways. Our data provide new insight into the mRNA and lncRNA cargos of teleost spermatozoa and uncover the possible involvement of novel endocrine mechanisms during the differentiation and maturation of spermatozoa

An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics and smFRET Identifies an Aggregation Mechanism

Protein aggregation is the underlying cause of many diseases, and also limits the usefulness of many natural and engineered proteins in biotechnology. Better mechanistic understanding and characterization of aggregation-prone states, is needed to guide protein engineering, formulation, and drug-targeting strategies that prevent aggregation. While several final aggregated states - notably amyloids - have been characterized structurally, very little is known about the native structural conformers that initiate aggregation. We used a novel combination of small-angle X-ray scattering (SAXS), atomistic molecular dynamics (MD) simulations, single-molecule FRET (smFRET), and aggregation-prone region (APR) predictions, to characterize structural changes in a native humanized Fab A33 antibody fragment, that correlated with the experimental aggregation kinetics. SAXS revealed increases in the native state radius of gyration, Rg, of 2.2% to 4.1%, at pH 5.5 and below, concomitant with accelerated aggregation. In a cutting-edge approach, we fitted the SAXS data to full molecular dynamics simulations from the same conditions, and located the conformational changes in the native state to the constant domain of the light chain (CL). This CL displacement was independently confirmed using smFRET measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted aggregation-prone region (APR), suggesting a likely mechanism through which they promote aggregation. Our findings provide a means by which aggregation-prone conformational states can be readily determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation