Boolean networks with robust and reliable trajectories

We construct and investigate Boolean networks that follow a given reliable trajectory in state space, which is insensitive to fluctuations in the updating schedule, and which is also robust against noise. Robustness is quantified as the probability that the dynamics return to the reliable trajectory after a perturbation of the state of a single node. In order to achieve high robustness, we navigate through the space of possible update functions by using an evolutionary algorithm. We constrain the networks to having the minimum number of connections required to obtain the reliable trajectory. Surprisingly, we find that robustness always reaches values close to 100 percent during the evolutionary optimization process. The set of update functions can be evolved such that it differs only slightly from that of networks that were not optimized with respect to robustness. The state space of the optimized networks is dominated by the basin of attraction of the reliable trajectory.Comment: 12 pages, 9 figure

Noise in random Boolean networks

We investigate the effect of noise on Random Boolean Networks. Noise is implemented as a probability $p$ that a node does not obey its deterministic update rule. We define two order parameters, the long-time average of the Hamming distance between a network with and without noise, and the average frozenness, which is a measure of the extent to which a node prefers one of the two Boolean states. We evaluate both order parameters as function of the noise strength, finding a smooth transition from deterministic ($p=0$) to fully stochastic ($p=1/2$) dynamics for networks with $K\le2$, and a first order transition at $p=0$ for $K>2$. Most of the results obtained by computer simulation are also derived analytically. The average Hamming distance can be evaluated using the annealed approximation. In order to obtain the distribution of frozenness as function of the noise strength, more sophisticated self-consistent calculations had to be performed. This distribution is a collection of delta peaks for K=1, and it has a fractal sructure for $K>1$, approaching a continuous distribution in the limit $K\gg1$.Comment: 9 pages, 8 figures, 1 tabl

Boolean networks with reliable dynamics

We investigated the properties of Boolean networks that follow a given reliable trajectory in state space. A reliable trajectory is defined as a sequence of states which is independent of the order in which the nodes are updated. We explored numerically the topology, the update functions, and the state space structure of these networks, which we constructed using a minimum number of links and the simplest update functions. We found that the clustering coefficient is larger than in random networks, and that the probability distribution of three-node motifs is similar to that found in gene regulation networks. Among the update functions, only a subset of all possible functions occur, and they can be classified according to their probability. More homogeneous functions occur more often, leading to a dominance of canalyzing functions. Finally, we studied the entire state space of the networks. We observed that with increasing systems size, fixed points become more dominant, moving the networks close to the frozen phase.Comment: 11 Pages, 15 figure

Activation of Kv7.4 in the cochlea as a therapeutic approach in a mouse model of age-reated hearing loss

Age-related hearing loss (ARHL) is the largest contributor to the substantial hearing loss prevalence and is recognized as a significant factor in psychological and medical morbidity. There is thus a tremendous need for a safe and effective pharmacological treatment. The loss of auditory hair cells, consisting of outer hair cells (OHCs) and inner hair cells (IHCs), has been described as the major cochlear pathology of ARHL. The OHC physiology depends largely on functional potassium ion (K+) recycling in the cochlea. One major component of the K+ recycling circuit is the voltage-gated potassium channel of subfamily q, member 1 (Kv7.1), which is localized in the stria vascularis (SV). Loss of KV7.1 has been associated with congenital deafness caused by impaired K+ secretion. Furthermore, OHC survival is linked to the voltage-gated potassium channel of subfamily q, member 4 (Kv7.4), which dominantly determines their membrane potential. Impaired surface expression of Kv7.4 leads to functional impairment and has also been associated with ARHL. Although multiple chemical Kv7 channel openers have been developed as a therapeutic approach, their applicability has not yet been demonstrated in an in vivo model. The aim of the present study was to investigate the effect of pharmacological Kv7.4 channel activation in the senescence-accelerated mouse prone strain 8 (SAMP8) model as a novel therapeutic approach against ARHL. Surprisingly, we observed a significant threshold variability in the auditory decline of SAMP8 mice over age. In search of an underlying cause, age- related OHC loss could not be linked to this threshold variability. However, an altered Kv7.4 expression in OHCs was linked to the SAMP8 auditory threshold variability over age, preceding OHC loss. Pharmacological Kv7.4 channel activation by novel, small- molecule Kv7.4 agonists was then investigated in SAMP8 mice using different routes of administration: ACOU085 was locally via transtympanic application and ACOU082 was systemically administered via topical application. We demonstrated that the locally administered ACOU085 readily diffused into the cochlea and was able to significantly reduce age-related auditory threshold shifts as well as OHC loss in SAMP8 mice. Our findings in an in-vivo mouse model suggest that pharmacological activation of Kv7.4 is a promising approach to prevent and decelerate age-related decline of auditory function and morphological OHC loss linked to ARHL

Exploring continuous and integrated strategies for the up- and downstream processing of human mesenchymal stem cells

The integration of up- and downstream unit operations can result in the elimination of hold steps, thus decreasing the footprint, and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC), where high numbers of pure cells, at low volumes, need to be delivered for therapy applications. The aim of this work is to perform a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. In particular, we have evaluated the impact of i) different operation modes (continuous and discontinuous) and ii) several TFF’s process parameters (e.g. membrane material and pore size, shear rate, permeate flux) have on cells’ expansion, recovery yield and quality (i.e. cell morphology, viability, identity and potency). Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio, and led to a shift in cells’ metabolism. Regarding TFF, the combination of polysulfone membranes with pore sizes higher than 0.45 μm with lower residence and processing times (translated by higher shear rates and permeate fluxes, respectively), led to the successful concentration of hMSC up to a factor of 20. The operation of TFF either in continuous or discontinuous allowed to concentrate cells with high cell recovery yields (\u3e80%) and viability (\u3e95%); furthermore, continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration allowed higher protein clearance (98%) with lower cell death, when comparing to discontinuous diafiltration. Overall, the integration of the optimized unit operations of concentration and washing led to a decrease in the bioprocess time in 40%, allowing for the recovery of over 70% of hMSC with high viability (\u3e95%), while maintaining cells’ morphology, immunophenotype, proliferation capacity and multipotent differentiation potential. This work shows that the integration of upstream and downstream unit operations is beneficial for cell therapy applications, where TFF showed to be an efficient methodology for the concentration and diafiltration of hMSC. The described process will have applicability to other stem cell types (e.g. human pluripotent stem cells) relevant for the cell therapy industry

Bioprocess engineering of insect cells for accelerating vaccines development

Insect cells emerged as a powerful and versatile platform for vaccines production, mostly using the lytic baculovirus expression vector system (BEVS). Stable expression in such hosts has been increasingly explored to circumvent BEVS-related drawbacks, but protein titers achieved to date are still seemingly low. The design of new or improved cell factories and bioprocess intensification strategies are therefore necessary to increase productivities and thus accelerate implementation of stable insect cell lines as a fast, cost-effective platform for vaccines manufacturing. In this work, we implemented an innovative site-specific recombination strategy based on flipase-mediated cassette exchange technology to establish reusable insect (Sf-9 and High Five) cell platforms for fast production of enveloped virus-like particles (VLPs). Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept demonstrated using two membrane proteins: the influenza HA protein (for vaccines) and the human beta-2 adrenergic receptor (for drug screening or antibody discovery). Aiming to improve production yields in developed stable cell lines, two bioprocess engineering schemes were evaluated (either individually or in combination): (i) adaptive laboratory evolution of insect cells to hypothermic culture conditions, and (ii) supplementation of insect cell cultures with productivity enhancers. The stable cell line expressing HIV Gag-VLPs was used as model. Under hypothermic culture conditions, adapted Sf-9 cells expressed up to 30-fold more HIV Gag-VLPs than non-adapted cells. Noteworthy, the element driving such increase in productivity is the adaptation process and not the temperature shift as the latter alone leads to lower production yields. A more modest increase in productivity (up to 7-fold) was observed when supplementing non-adapted cell cultures with productivity enhancers NaBu and DMSO. No synergistic effect was observed when combining adapted cells and supplementation with productivity enhancers. Production of HIV Gag-VLPs was successfully scaled-up to stirred-tank bioreactors. The adapted cell line was then pseudo-typed with influenza HA protein for production of Gag-HA VLPs, and their performance benchmarked against (i) parental Sf-9 cells stably expressing Gag-HA VLPs and (ii) insect cells-BEVS, both cultured under standard temperature conditions (27C). Adapted cells showed increased production of Gag-HA VLPs when compared to parental/stable cells, corroborating previously obtained data, but still lower when compared to insect cells-BEVS. Bioprocess intensification strategies are currently under in-house testing to further improve yields of adapted cells and thus shorten the gap between stable insect cells and IC-BEVS. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist and accelerate vaccines development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBB-BIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

Multi-gene phylogeny and divergence estimations for Evaniidae (Hymenoptera)

Ensign wasps (Hymenoptera: Evaniidae) develop as predators of cockroach eggs (Blattodea), have a wide distribution and exhibit numerous interesting biological phenomena. The taxonomy of this lineage has been the subject of several recent, intensive efforts, but the lineage lacked a robust phylogeny. In this paper we present a new phylogeny, based on increased taxonomic sampling and data from six molecular markers (mitochondrial 16S and COI, and nuclear markers 28S, RPS23, CAD, and AM2), the latter used for the first time in phylogenetic reconstruction. Our intent is to provide a robust phylogeny that will stabilize and facilitate revision of the higher-level classification. We also show the continued utility of molecular motifs, especially the presence of an intron in the RPS23 fragments of certain taxa, to diagnose evaniid clades and assist with taxonomic classification. Furthermore, we estimate divergence times among evaniid lineages for the first time, using multiple fossil calibrations. Evaniidae radiated primarily in the Early Cretaceous (134.1–141.1 Mya), with and most extant genera diverging near the K-T boundary. The estimated phylogeny reveals a more robust topology than previous efforts, with the recovery of more monophyletic taxa and better higher-level resolution. The results facilitate a change in ensign wasp taxonomy, with Parevania, and Papatuka, syn. nov. becoming junior synonyms of Zeuxevania, and Acanthinevania, syn. nov. being designated as junior synonym of Szepligetella. We transfer 30 species to Zeuxevania, either reestablishing past combinations or as new combinations. We also transfer 20 species from Acanthinevania to Szepligetella as new combinations

O atendimento pré-hospitalar prestados por leigos a vítimas de acidentes de trânsito terrestre: revisão integrativa / Pre-hospital care provided by laypeople to victims of road traffic accidents: an integrative review

Anualmente, cerca de 1,3 milhões de pessoas têm as suas vidas interrompidas devido a acidentes de trânsitos terrestres (ATT). Ainda que sejam observadas altas prevalências, foi observado uma queda anual de 7% do número de óbitos por ATT no Brasil entre os anos 2015 e 2019. O Ministério da Saúde (MS) atribui a esta queda como consequência da intensificação de ações de educação no trânsito. Outro fator de extrema relevância na redução da morbimortalidade das vítimas de lesões produzidas por acidentes é o atendimento adequado. Sendo assim, esta Revisão Integrativa (RI) tem como objetivo identificar as principais características que envolvem o treinamento de pessoas leigas no atendimento ao ATT. Este trabalho trata-se de uma Revisão Integrativa de Literatura (RI). Os resultados dos estudos que compuseram essa Revisão Integrativa evidenciaram que o treinamento de pessoas leigas, a fim de capacitá-las para o atendimento de vítimas de acidentes de trânsito terrestre, se comportou como uma importante ferramenta na redução desses agravos

NURSING CARE IN THE PROMOTION OF FAMILY CARE FOR THE NEWBORN PREMATURE

Objective: To reflect on nurse's role in promoting family care in the premature neonate from the analysis of scientific production of nursing nationally and internationally on this topic. Methods: This is a literature review on nursing care in the promotion of family care for the newborn infants. Data were published in the last ten years, in journals indexed in databases: LILACS and BDENF. We found 72 publications of which 7 fell on the goal of the research. For analysis of the productions were analyzed to content in the thematic mode, resulting in 3 categories: a) Nurses in Interaction and Parental Guidance b) Care of premature children c) The family and family support network. Results: There were that the nursing team, is still organizing to get the parents in the NICU. Conclusion: Thus, the publications emphasize the importance of not only spur the team interaction and parental guidance, but also to introduce dynamic with professionals and parents as well as support groups among parents who are experiencing the process of prematurity the child and parents who have lived through this process stronger and stronger reception and exchange of experiences. Descriptors: Prematurity, Care, Nursing, Family.Objetivo: Refletir sobre atuação do enfermeiro na promoção do cuidado familiar ao neonato prematuro a partir da análise da produção científica da enfermagem nacional e internacional acerca desta temática. Métodos: Trata-se de uma revisão bibliográfica sobre a assistência de enfermagem na promoção do cuidado familiar ao neonato prematuro. Os dados analisados foram publicações dos últimos dez anos, em revistas indexadas nas bases de dados: LILACS e BDENF. Foram encontradas 72 publicações das quais 7 se enquadravam no objetivo da pesquisa. Para análise das produções foi adotada a análise de conteúdo na modalidade temática, originando 3 categorias: a) O enfermeiro na Interação e Orientação Parental; b) Cuidado ao filho prematuro; c) A família e a rede de apoio familiar. Resultados: identificamos que a equipe de enfermagem, ainda esta se organizando para receber os pais na UTIN. Conclusão: Dessa forma, as publicações enfatizam a importância de não só estimularmos a equipe na interação e orientação dos pais, mas também de se introduzir dinâmicas com os profissionais e pais, bem como dos grupos de apoio entre pais que estão vivendo o processo de prematuridade do filho e de pais que já viveram esse processo, fortalecendo cada vez mais o acolhimento e a troca de experiências. Descritores: Prematuridade, Cuidados, Enfermagem, Família

Roles of non-coding RNA in sugarcane-microbe interaction

Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs) in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae. Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs) were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a coppertransporter gene whose expression was induced in the presence of A. avenae, while the siRNAs were repressed in the presence of A. avenae. Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR). Among these miRNAs, miR408—a copper- microRNA—was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 50RACE (rapid amplification of cDNA ends) assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly