Binding affinities and activation of Asp712Ala and Cys100Ser mutated kinin B1 receptor forms suggest a bimodal scheme for the molecule of bound-DABK

AbstractMutant forms of kinin B1 receptor (B1R) and analogs of the full agonist des-Arg9-bradykinin (DABK) were investigated aiming to verify the importance of selected receptor residues and of each agonist-peptide residue in the specific binding and activation. Linked by a specific disulfide bond (Cys100–Cys650), the N-terminal (Nt) and the EC3 loop C-terminal (Ct) segments of angiotensin II (AngII) receptor 1 (AT1R) have been identified to form an extracellular site for binding the agonist Nt segment (Asp1 and Arg2 residues). Asp712 residue at the receptor EC3 loop binds the peptide Arg2 residue. By homology, a similar site might be considered for DABK binding to B1R since this receptor contains the same structural elements for composing the site in AT1R, namely the disulfide bond and the EC3 loop Asp712 residue. DABK, Alan-DABK analogs (n=Ala1-, Ala2-, Ala3-, Ala4-, Ala5-, Ala6-, Ala7-, Ala8-DABK), and other analogs were selected to binding wild-type, Asp712Ala and Cys100Ser mutated B1R receptors. The results obtained suggested that the same bimodal scheme adopted for AngII-AT1R system may be applied to DABK binding to B1R. The most crucial similarity in the two cases is that the Nt segments of peptides equally bind to the homologous Asp712 residue of both AT1R and B1R extracellular sites. Confirming this preliminary supposition, mutation of residues located at the B1R extracellular site as EC3 loop Asp712 and Cys100 caused the same modifications in biological assays observed in AT1R submitted to homologous mutations, such as significant weakening of agonist binding and reduction of post-receptor-activation processes. These findings provided enough support for defining a site that determines the specific binding of DABK to B1R receptors

Human pluripotent stem cell expansion in vertical-wheel bioreactors

Human induced pluripotent stem cells (hiPSC) have been regarded as an enormous breakthrough for medicine, since they can be derived from patients and be used to generate virtually all types of cells in the human body. One of the great bottlenecks in the usage of these cells for regenerative medicine or drug discovery applications is their expansion to relevant quantities. The Vertical-Wheel Bioreactors (PBS Biotech) present a novel scalable bioreactor configuration, whose agitation mechanism allows for homogeneous mixing conditions inside the single-use vessel, while conveying less shear stress to the cells when compared to traditional alternatives. These characteristics are advantageous for hiPSC expansion and thus, in this work, hiPSC were expanded in the Vertical-Wheel Bioreactor using different strategies, namely culturing the cells 1) on microcarriers and 2) as floating aggregates. In the first approach, cells were cultured under xeno-free conditions, using the Essential 8 medium together with microcarriers and coatings devoid of any animal-derived products [1]. The culture conditions were optimized in terms of initial cell/microcarrier ratio, inoculation method and agitation rate, in the PBS 0.1 vessel (working volume: 80 mL). The cells were successfully expanded, maintaining a normal karyotype, up to a 6.7-fold increase in cell number, after 6 days. These optimized culture conditions were successfully repeated in a larger vessel, the PBS 0.5 (300 mL working volume) demonstrating the scalability of the Vertical-Wheel system. In the second approach, hiPSC were expanded as floating aggregates, a methodology which does not require a separation step at the end of culture, to remove microcarriers, facilitating the downstream processing and Good Manufacturing Practice-compliance of the process. Cells were cultured in the PBS 0.1 (working volume: 60 mL), using mTeSR1, a serum-free medium and were monitored throughout culture regarding growth kinetics, aggregate size distribution and expression of pluripotency markers. The Vertical-Wheel Bioreactors were shown to efficiently keep the cell aggregates in suspension, under lower linear agitation speeds than an equivalent volume spinner flask (7 cm/s vs. 13 cm/s). Following 7 days of culture, cells were expanded up to a 5.2 ± 0.6-fold increase in cell number. The hiPSC aggregates increased in size over time, from an average diameter of 135 ± 61 µm to 397 ± 119 µm after 7 days. Pluripotency was maintained throughout time, as assessed by sustained high (\u3e 80%) expression of pluripotency markers OCT4, SOX2 and TRA-1-60, and low (\u3c 10%) expression of early differentiation marker SSEA-1. The results were validated using a second hiPSC line. This study revealed that the Vertical-Wheel Bioreactor allows hiPSC growth either on microcarriers and as aggregates and suggested it to have advantages versus other configurations. These results make the Vertical-Wheel Bioreactor a promising platform for hiPSC expansion and, prospectively, differentiation approaches, contributing for the generation of bona fide cells for various biomedical applications, namely drug screening, disease modelling, and, ultimately, for Regenerative Medicine. [1] Rodrigues CAV, Silva TP, Nogueira DES, Fernandes TG, Hashimura Y, Wesselschmidt R, Diogo MM, Lee B, Cabral JMS (2018), “Scalable Culture Of Human Induced Pluripotent Cells On Microcarriers Under Xeno‐Free Conditions Using Single‐Use Vertical‐Wheel™ Bioreactors”, Journal of Chemical Technology and Biotechnology, DOI: 10.1002/jctb.573

Sodium reduction in traditional dry-cured pork belly using glasswort powder (Salicornia herbacea) as a partial NaCl replacer

Sodium chloride (NaCl) is a key ingredient in the processing of traditional dry-cured meat products by improving microbial safety, sensory attributes and technological properties. However, increasing concern about the consumption of sodium and health has been supporting the development of low-sodium meat products. Several strategies to reduce sodium in dry-cured meat product have been tested, although the followed approaches sometimes result in undesirable characteristics concerning flavor, texture and mouthfeel. The use of halophytic plants such as glasswort (Salicornia herbacea) in food matrices has been suggested as a novel strategy to reduce sodium content, due its salty flavor. The main aim of the present study is to produce traditional dry-cured pork bellies from the Bísaro breed using glasswort as a NaCl partial replacer, and compare it with dry-cured bellies salted either with NaCl or a mix of NaCl + KCl. Control bellies (BC) were salted with 100% of NaCl, the second formulation (BK) had 50% of NaCl and 50% of KCl, and the third formulation (BG) had 90% of NaCl and 10% of glasswort powder (GP). After production, the bellies were evaluated for aw, pH, CIELab coordinates, weight loss, proximal composition, TBARS, collagen and chloride contents, fatty acid profile and sensory attributes. The use of BG in dry-cured pork bellies did not affect processing indicators such as weight loss, aw and pH. Concerning CIELab, only the coordinates L* and hue angle from the external surface color of BG were statistically different from BC and BK. As expected, ash and NaCl contents differed from BG to the other two formulations. SFA and indexes AI and TI were lower, whereas the MUFA and h/H ratio were higher in BG than other treatments, leading to a product with a healthier lipid profile. The sensory evaluation revealed differences in appearance, taste and flavor among treatments, but did not indicate any negative effects of BG in the product attributes. This study reinforces the potential of BG as a natural sodium reducer for the production of traditional dry-cured pork belliesThe authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2021). Grants of I.F., A.L. and L.V. are due to NORTE-01-0247-FEDER-072234. P.E.S.M. acknowledges postdoctoral fellowship support from the Ministry of Science and Innovation (MCIN, Spain) “Juan de la Cierva” program (IJC2020-043358-I).info:eu-repo/semantics/publishedVersio

New entomological and virological data on the vectors of sylvatic yellow fever in Brazil

Institut Français de Recherche Scientifique pour le Développement en Coopération. Belém, PA, Brasil / Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.O presente trabalho enfatiza resultados recentes sobre eco-epidemiologia da febre amarela, obtidos em duas localidades ecologicamente muito diferentes: Barcarena (PA), situada na floresta amazônica densa, e a região de Campo Grande (MS), coberta de cerrado atravessado por florestas de galeria na região Centro-oeste. No primeiro local foi isolada uma amostra a partir de um lote de 6 Haemagogus janthinomys. Na região de Campo Grande, 2. 480 mosquitos antropófilos foram coletados, dos quais cerca de 40% eram vetores potenciais da FA. Essas espécies, classificadas por ordem decrescente de abundãncia, eram: Ae. scapularis, Sa. chloropterus, Hg. janthinomys, Hg. leucocelaenus, Hg. spegazinii e Sa. soperi. Quatro amostras de FA foram isolados a partir de Hg. janthinomys, uma de Sa. chloropterus (primeiro registro na América do Sul), e uma de Sa. soperi (primeiro registro). As taxas mínimas de infecção variaram segundo os locais coleta, mas foram sempre alta comparação com dados anteriores. A taxa média de sobrevivência diária do Hg. janthinomys é igual a 0,9635, permitindo estimar o seu valor na ocasião em que os caso humanos mais recentes foram provavelmente contaminados. deduziu-se que as epizootias foram muito intensas e mais ou menos simultâneas nos dois locais considerados. O maior problema a ser resolvido é o modo de re-introdução do vírus, ou sua sobreviv~encia, em cada região sob estudo

Дискусійні питання історії ЗУНР

У статті аналізуються питання, які все ще залишаються в історіографії Західноукраїнської Народної Республіки. Автор звертає увагу на контроверсійні точки зору щодо революційних подій, які відбулися 1 листопада 1918 р. у Львові, стосовно часу існування ЗУНР, Акту злуки УНР та ЗУНР, причин поразки Української Галицької армії тощо.The author of the article analyses the questions that are still disputable in modern Ukrainian historiography. The author attracts our attention to the controversial points of view concerning the type of revolutionary events which took place on the 1 st of November 1918 in Lviv, concerning the time of WUPR existence, concerning the Act of Unification of Western Ukrainian People’s Republic end Ukrainian People’s Republic , concerning the reasons of the defeat of Ukrainian army, etc

Production of fructo-oligosaccharides by Aspergillus ibericus and their chemical characterization

A great demand for prebiotics is driving the search for new sources of fructo-oligosaccharides (FOS) producers and for FOS with differentiated functionalities. In the present work, FOS production by a new isolated strain of Aspergillus ibericus was evaluated. The temperature of fermentation and initial pH were optimized in shaken flask to yield a maximal FOS production, through a central composite experimental design. FOS were produced in a one-step bioprocess using the whole cells of the microorganism. The model (R2 = 0.918) predicted a yield of 0.56, experimentally 0.53 ± 0.03 gFOS.ginitial sucrose1 was obtained (37.0 °C and a pH of 6.2). A yield of 0.64 ± 0.02 gFOS.ginitial sucrose1 was obtained in the bioreactor, at 38 h, with a content of 118 ± 4 g.L1 in FOS and a purity of 56 ± 3%. The chemical structure of the FOS produced by A. ibericus was determined by HPLC and NMR. FOS were identified as 1-kestose, nystose, and 1F-fructofuranosylnystose. In conclusion, A. ibericus was found to be a good alternative FOS producer.Clarisse Nobre acknowledges the Portuguese Foundation forScience and Technology (FCT) for her Post-Doc Grant [ref. SFRH/BPD/87498/ 2012] and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124FEDER-027462), the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation(NORTE-01-0145-FEDER-000004) and the project MultiBiorefinery (POCI-01-0145-FEDER-016403) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Raman spectroscopy vs voltammetry: a voltammetric approach to elucidate different chemicals in a range of pharmaceutical tablets.

Over the years, Electroanalysis has been widely applied to elucidate redox behavior of novel molecules. The selectivity and low cost are spotlight features in pharmacopeial methods of identification, that can be reached by voltammetric approaches. In this work, differential pulse voltammetric (DPV) profile and the slope of the linear regression obtained from calibration graphs along with the scan study are proposed as new perspective of identification assays. With the proposed methodology we were able to identify the similarities among DPV profile and the slopes obtained for each tablet. In addition, this new technology was successfully employed to identify the following chemicals: Paracetamol (PAR), Promethazine (PMZ), Diclofenac (DIC), Piroxicam (PRX), Indomethacin (IND) and Cyclobenzaprine (CBP) in pharmaceutical assays using Pencil Graphite Electrodes. Furthermore, our new methodology was effectively compared to Raman Spectroscopy for the analysis of the range of chemicals in the pharmaceutical assays