Low cost microfluidic device for partial cell separation: micromilling approach

Several studies have already demonstrated that it is possible to perform blood flow studies in microfluidic systems fabricated by using low-cost techniques. However, most of these techniques do not produce microchannels smaller than 100 microns and as a result they have several limitations related to blood cell separation. Recently, manufacturers have been able to produce milling tools smaller than 100 microns, which consequently have promoted the ability of micromilling machines to fabricate microfluidic devices able to perform separation of red blood cells (RBCs) from plasma. In this work, we show the ability of a micromilling machine to manufacture microchannels with dimensions down to 30 microns. Additionally, we show for the first time the ability of the proposed microfluidic device to enhance the cell-free layer close to the walls, leading to perform partial separation of RBCs from plasma.The authors acknowledge the financial support provided by PTDC/SAU-ENB/116929/2010 and EXPL/EMSSIS/ 2215/2013 from FCT (Science and Technology Foundation), COMPETE, QREN and European Union (FEDER). RR and DP acknowledge, respectively, the PhD scholarships SFRH/BD/97658/2013 and SFRH/BD/89077/2012 attributed by FCT

In vitro blood flow and cell-free layer in hyperbolic microchannels: visualizations and measurments

Red blood cells (RBCs) in microchannels has tendency to undergo axial migration due to the parabolic velocity profile, which results in a high shear stress around wall that forces the RBC to move towards the centre induced by the tank treading motion of the RBC membrane. As a result there is a formation of a cell free layer (CFL) with extremely low concentration of cells. Based on this phenomenon, several works have proposed microfluidic designs to separate the suspending physiological fluid from whole in vitro blood. This study aims to characterize the CFL in hyperbolic-shaped microchannels to separate RBCs from plasma. For this purpose, we have investigated the effect of hyperbolic contractions on the CFL by using not only different Hencky strains but also varying the series of contractions. The results show that the hyperbolic contractions with a Hencky strain of 3 and higher, substantially increase the CFL downstream of the contraction region in contrast with the microchannels with a Hencky strain of 2, where the effect is insignificant. Although, the highest CFL thickness occur at microchannels with a Hencky strain of 3.6 and 4.2 the experiments have also shown that cells blockage are more likely to occur at this kind of microchannels. Hence, the most appropriate hyperbolic-shaped microchannels to separate RBCs from plasma is the one with a Hencky strain of 3.The authors acknowledge the financial support provided by PTDC/SAU-ENB/116929/2010 and EXPL/EMS-SIS/2215/2013 from FCT (Fundação para a Ciência e a Tecnologia), COMPETE, QREN and European Union (FEDER). R.O. Rodrigues, D. Pinho and P. C. Sousa acknowledge the scholarships SFRH/BD/97658/2013, SFRH/BD/89077/2012 and SFRH/BPD/75258/2010, respectively, all attributed by FCT

Essential genetic findings in neurodevelopmental disorders

Neurodevelopmental disorders (NDDs) represent a growing medical challenge in modern societies. Ever-increasing sophisticated diagnostic tools have been continuously revealing a remarkably complex architecture that embraces genetic mutations of distinct types (chromosomal rearrangements, copy number variants, small indels, and nucleotide substitutions) with distinct frequencies in the population (common, rare, de novo). Such a network of interacting players creates difficulties in establishing rigorous genotype-phenotype correlations. Furthermore, individual lifestyles may also contribute to the severity of the symptoms fueling a large spectrum of gene-environment interactions that have a key role on the relationships between genotypes and phenotypes.Herein, a review of the genetic discoveries related to NDDs is presented with the aim to provide useful general information for the medical community.info:eu-repo/semantics/publishedVersio

Variations in salivary function in a rodent model of pre-diabetes

Diabetes is a widespread disease representing an enormous part of the total health costs. An early diagnostic could be of extremely importance both for the understanding and prevention of this pathology. Saliva is a fluid with increasing interest as a source of biomarkers for disease diagnostic and saliva protein composition changes have already been reported for diabetic individuals. However, the studies were performed after the onset of the disease and it is unknown if salivary changes are present in the early stages of development of the disease or a characteristic of overt diabetes. Wistar rats have been selected for their glucose intolerance (GIR). GIR females were compared with Wistar females with normal glucose tolerance (control) for changes in saliva protein composition and salivary gland histology. Fasting glycemias were observed to be normal (<95 mg/dl) in GIR animals, indicating an absence of a diabetic state. However they presented an abnormal increase in glycemia after a glucose bolus. For salivary parameters a marked increase in total protein concentration and alpha-amylase activity occurred in GIR animals, comparatively to controls. After separation of salivary proteins by SDS PAGE differences between the experimental groups for some protein bands, with apparent molecular masses ranging from 20 to 55 kDa were observed. Different expression of alphaamylase at salivary gland duct level is also apparent for pre-diabetic animals. Although preliminary, these results suggest changes in saliva occurring before the onset of diabetes, reinforcing the interest of further investigation of saliva composition for the diagnostic of pre-diabetic condition, ultimately allowing an early intervention and eventually the prevention of disease development

Caracterização de materiais compósitos à base de gesso FGD

Este estudo visa a valorização de diversos subprodutos industriais como o gesso da dessulfuração de gases de combustão das centrais termoeléctricas, designado convencionalmente por gesso FGD (“flue gas desulfurization”), o re-granulado de cortiça resultante do fabrico de placas de aglomerado negro de cortiça e as fibras têxteis resultantes da reciclagem de pneus usados. Os materiais compósitos resultantes das misturas destes subprodutos podem ser conformados por dois processos distintos, a moldagem e a prensagem, obtendo-se produtos com características distintas. Por moldagem obtêm-se um compósito leve e por prensagem obtêm-se um material mais denso, com maiores resistências mecânicas e melhor acabamento superficial. Estes compósitos poderão ter várias aplicações na construção, nomeadamente no fabrico de blocos para paredes interiores dos edifícios. Neste contexto, foi realizada uma campanha experimental de modo a determinar as características mecânicas dos referidos compósitos no sentido de validar a sua aplicabilidade na construção

Caracterização de materiais compósitos à base de gesso FGD

Este estudo visa a valorização de diversos subprodutos industriais como o gesso da dessulfuração de gases de combustão das centrais termoeléctricas, designado convencionalmente por gesso FGD (“flue gas desulfurization”), o re-granulado de cortiça resultante do fabrico de placas de aglomerado negro de cortiça e as fibras têxteis resultantes da reciclagem de pneus usados. Os materiais compósitos resultantes das misturas destes subprodutos podem ser conformados por dois processos distintos, a moldagem e a prensagem, obtendo-se produtos com características distintas. Por moldagem obtêm-se um compósito leve e por prensagem obtêm se um material mais denso, com maiores resistências mecânicas e melhor acabamento superficial. Estes compósitos poderão ter várias aplicações na construção, nomeadamente no fabrico de blocos para paredes interiores dos edifícios. Neste contexto, foi realizada uma campanha experimental de modo a determinar as características mecânicas dos referidos compósitos no sentido de validar a sua aplicabilidade na construção

Applying reduce SNP assays for inferring C-lineage introgression patterns in Iberian honeybee populations of the Azores archipelago

The genetic composition of the honeybee populations of the Macaronesian archipelago of the Azores is poorly known. Until now, only honeybee populations of the island of São Miguel have been surveyed for genetic variation through the use of the tRNAleu-cox2 intergenic mitochondrial DNA region and microsatellites. Here, we combine data from the mtDNA obtained with the DraI test (intergenic region) and from the nuclear DNA obtained with newly developed reduced SNP assays to provide a complete picture of introgression patterns in the Azorean honeybee populations at both mitochondrial and nuclear compartments. The sampling was carried out in 2014 and 2015 and comprised 474 colonies widely distributed across the 8 islands populated by honeybees. Our cyto-nuclear results show that C-derived introgression varies across the archipelago ranging from virtually pure populations of the Iberian honeybee in the island of Santa Maria (Q-values 30%). The introgression levels are alarming and contrast with those of the Iberian honeybee populations of the mainland in Iberia, which are still virtually free of C-derived introgression, despite frequent importation of commercial queens.info:eu-repo/semantics/publishedVersio

Human phenylalanine hydroxylase as the case study

Funding Information: Authors acknowledge Sofarimex, Indústria Química e Farmacêutica SA, Portugal, for all the support concerning freeze-drying studies. This work was supported by FEDER and Fundação para a Ciência e a Tecnologia, I. P. through iMED.ULisboa (Projects UIDB/04138/2020 and UIDP/04138/2020), iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and research project PTDC/EBB-BIO/101237/2008 and research grant SFRH/BD/47946/2008 (to Paulo R. Lino). This work has also received funding from the National PKU Alliance, USA. The authors would like to thank Luís Miguel Ramos and Cátia Nascimento who contributed to the exploratory research that culminated in the work herein presented. Funding Information: Authors acknowledge Sofarimex, Indústria Química e Farmacêutica SA, Portugal, for all the support concerning freeze-drying studies. This work was supported by FEDER and Fundação para a Ciência e a Tecnologia, I. P. through iMED.ULisboa (Projects UIDB/04138/2020 and UIDP/04138/2020), iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and research project PTDC/EBB-BIO/101237/2008 and research grant SFRH/BD/47946/2008 (to Paulo R. Lino). This work has also received funding from the National PKU Alliance, USA. Publisher Copyright: © 2023 The Author(s)The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.publishersversionpublishe

Retention of fatty acyl desaturase 1 (fads1) in Elopomorpha and Cyclostomata provides novel insights into the evolution of long-chain polyunsaturated fatty acid biosynthesis in vertebrates

Background Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C18 precursors through consecutive elongations and desaturations. It has been postulated that the abundance of LC-PUFA in the marine environment has remarkably modulated the gene complement and function of Fads in marine teleosts. In vertebrates two fatty acyl desaturases, namely Fads1 and Fads2, encode ∆5 and ∆6 desaturases, respectively. To fully clarify the evolutionary history of LC-PUFA biosynthesis in vertebrates, we investigated the gene repertoire and function of Fads from species placed at key evolutionary nodes. Results We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered. Conclusions We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes